U.S. patent application number 10/553869 was filed with the patent office on 2006-09-07 for cleavage of fusion proteins using granzyme b protease.
Invention is credited to Charlotte Harkjaer Fynbo, Rikke Hoegh Lorentsen.
Application Number | 20060199251 10/553869 |
Document ID | / |
Family ID | 36806138 |
Filed Date | 2006-09-07 |
United States Patent
Application |
20060199251 |
Kind Code |
A1 |
Lorentsen; Rikke Hoegh ; et
al. |
September 7, 2006 |
Cleavage of fusion proteins using granzyme b protease
Abstract
A method for the preparation of a polypeptide of interest in
authentic form by enzymatic cleavage of fusion proteins using
Granzyme B protease (EC 3.4.21.79). There is also provided fusion
proteins comprising a polypeptide of interest and a fusion partner,
wherein the junction region between the polypeptide of interest and
the fusion partner comprises a Granzyme B protease cleavage site
adjacent to the polypeptide of interest, and a human Granzyme B
protease variant wherein the Cystein residue no. 228
(chymotrypsinogen numbering) is mutated to Phenylalanine.
Inventors: |
Lorentsen; Rikke Hoegh;
(Frederiksberg, DK) ; Fynbo; Charlotte Harkjaer;
(Horsholm, DK) |
Correspondence
Address: |
HUNTON & WILLIAMS LLP;INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W.
SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Family ID: |
36806138 |
Appl. No.: |
10/553869 |
Filed: |
April 23, 2004 |
PCT Filed: |
April 23, 2004 |
PCT NO: |
PCT/DK04/00282 |
371 Date: |
October 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60464663 |
Apr 23, 2003 |
|
|
|
Current U.S.
Class: |
435/69.7 ;
435/226; 435/320.1; 435/325; 536/23.2 |
Current CPC
Class: |
C07K 2319/50 20130101;
C12N 9/6467 20130101; C12N 15/62 20130101 |
Class at
Publication: |
435/069.7 ;
435/226; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C07H 21/04 20060101
C07H021/04; C12P 21/04 20060101 C12P021/04; C12N 9/64 20060101
C12N009/64 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 23, 2003 |
DK |
PA 2003-00616 |
Claims
1. A method for the preparation of a polypeptide of interest in
authentic form, said method comprising the steps of: (i) providing
a fusion protein comprising, from its N-terminal to its C-terminal,
(a) a fusion partner, (b) a Granzyme B protease recognition site
comprising a Granzyme B protease cleavage site, (c) a polypeptide
of interest, wherein said cleavage site is adjacent to the
polypeptide of interest, and (ii) contacting said fusion protein
with Granzyme B protease to cleave it at said cleavage site to
yield said polypeptide of interest in authentic form.
2. A method according to claim 1, wherein the Granzyme B protease
recognition site has an amino acid sequence of the general formula:
P4 P3 P2 P1.dwnarw. wherein P4 is amino acid I or V P3 is amino
acid E, Q or M P2 is X, where X denotes any amino acid, P1 is amino
acid D, and .dwnarw. is said Granzyme B protease cleavage site.
3. A method according to claim 1, wherein the Granzyme B protease
recognition site has an amino acid sequence selected from the group
consisting of ICPD.dwnarw., IEAD.dwnarw., IEPD.dwnarw.,
IETD.dwnarw., IQAD.dwnarw., ISAD.dwnarw., ISSD.dwnarw.,
ITPD.dwnarw., VAPD.dwnarw., VATD.dwnarw., VCTD.dwnarw.,
VDPD.dwnarw., VDSD.dwnarw., VEKD.dwnarw., VEQD.dwnarw.,
VGPD.dwnarw., VEID.dwnarw., VRPD.dwnarw., VTPD.dwnarw.,
LEED.dwnarw., LEID.dwnarw., LGND.dwnarw., LGPD.dwnarw., and
AQPD.dwnarw., and wherein .dwnarw. is said Granzyme B protease
cleavage site.
4. A method according to claim 2, wherein the general formula
furthermore comprises the amino acids P1' and P2' resulting in the
general formula P4 P3 P2 P1.dwnarw.P1.dwnarw.P2', wherein P1' is X
where X denotes any amino acid, P2' is G, and wherein P1' and P2'
is a part of the polypeptide of interest.
5. A method according to claim 2, wherein the general formula
furthermore comprises the amino acids P1', P2', P3' and P4'
resulting in the general formula P4 P3 P2 P1.dwnarw.P1'P2'P3'P4',
wherein P4' is D or E, and wherein P1', P2', P3' and P4' is a part
of the polypeptide of interest.
6. A method according to claim 1, wherein the polypeptide of
interest is selected from the group consisting of an enzyme, a
polypeptide hormone, a single chain antibody variable region
fragment, and apolipoprotein A.
7. A method according to claim 6, wherein the polypeptide hormone
is selected from the group consisting of somatotrophin, glucagon,
insulin and inteferon.
8. A method according to claim 6, wherein the enzyme is Granzyme
B.
9. A method according to claim 1, wherein the fusion partner is an
affinity-tag.
10. A method according to claim 9, wherein the affinity-tag is
selected from the group consisting of a polyhistidine-tag, a
polyarginine-tag, a FLAG-tag, a Strep-tag, a c-myc-tag, a S-tag, a
calmodulin-binding peptide, a cellulose-binding peptide, a
chitin-binding domain, a glutathione S-transferase-tag, and a
maltose binding protein.
11. A method according to claim 1, wherein the Granzyme B protease
is selected from the group consisting of human Granzyme B protease,
mouse Granzyme B protease and rat Granzyme B protease.
12. A method according to claim 11, wherein the Granzyme B protease
is a human Granzyme B protease variant as shown in SEQ ID NO 57,
wherein the Cystein residue no. 228 (chymotrypsinogen numbering) is
mutated to Phenylalanine.
13. A method according to claim 1, wherein the Granzyme B protease
is in an immobilised form.
14. A method according to claim 13, wherein the Granzyme B protease
is immobilised via the C-terminus.
15. A method according to claim 13, wherein the Granzyme B protease
is immobilised via a lysine amino acid residue.
16. A method according to claim 10, wherein the affinity-tag is a
polyhistidine-tag, and wherein the fusion protein is contacted with
said Granzyme B protease in the presence of Ni.sup.2+ ions and
Nitrilotriacetic Acid (NTA).
17. A method according to claim 15, wherein the concentration of
Ni.sup.2+ is in the range of 1-20 mM, and the concentration of NTA
is in the range of 1-20 mM.
18. A fusion protein comprising, from its N-terminal to its
C-terminal, (a) a fusion partner, (b) a Granzyme B protease
recognition site comprising a Granzyme B protease cleavage site,
and (c) a polypeptide of interest, wherein said cleavage site is
adjacent to the polypeptide of interest.
19. A fusion protein according to claim 18, wherein the Granzyme B
protease recognition site has an amino acid sequence of the general
formula: P4 P3 P2 P1.dwnarw. wherein P4 is amino acid I or V P3 is
amino acid E, Q or M P2 is X, where X denotes any amino acid, P1 is
amino acid D, and .dwnarw. is said Granzyme B protease cleavage
site.
20. A fusion protein according to claim 18, wherein the Granzyme B
protease recognition site has an amino acid sequence selected from
the group consisting of ICPD.dwnarw., IEAD.dwnarw., IEPD.dwnarw.,
IETD.dwnarw., IQAD.dwnarw., ISAD.dwnarw., ISSD.dwnarw.,
ITPD.dwnarw., VAPD.dwnarw., VATD.dwnarw., VCTD.dwnarw.,
VDPD.dwnarw., VDSD.dwnarw., VEKD.dwnarw., VEQD.dwnarw.,
VGPD.dwnarw., VEID.dwnarw., VRPD.dwnarw., VTPD.dwnarw.,
LEED.dwnarw., LEID.dwnarw., LGND.dwnarw., LGPD.dwnarw., and
AQPD.dwnarw., and wherein .dwnarw. is said Granzyme B protease
cleavage site.
21. A fusion protein according to claim 19, wherein the general
formula furthermore comprises the amino acids P1' and P2' resulting
in the general formula P4 P3 P2 P1.dwnarw.P1'P2', wherein P1' is X
where X denotes any amino acid, P2' is G, and wherein P1' and P2'
is a part of the polypeptide of interest.
22. A fusion protein according to claim 19, wherein the general
formula furthermore comprises the amino acids P1', P2', P3' and P4'
resulting in the general formula P4 P3 P2 P1.dwnarw.P1'P2'P3'P4',
wherein P4' is D or E, and wherein P1', P2', P3' and P4' is a part
of the polypeptide of interest.
23. A fusion protein according to claim 18, wherein the polypeptide
of interest is selected from the group consisting of an enzyme, a
polypeptide hormone, a single chain antibody variable region
fragment, and apolipoprotein A.
24. A fusion protein according to claim 23, wherein the polypeptide
hormone is selected from the group consisting of somatotrophin,
glucagon, insulin and inteferon.
25. A fusion protein according to claim 23, wherein the enzyme is
Granzyme B.
26. A fusion protein according to claim 25, wherein Granzyme B
comprises a C-- terminal polyhistidine-tag.
27. A fusion protein according to claim 25, selected from the group
consisting of pro-IEPD-GrB--H6 (SEQ ID NO 2) and pro-IEAD-GrB--H6
(SEQ ID NO 3).
28. A fusion protein according to claim 25, selected from the group
consisting of pro-IEPD-GrB--H6 C228A (SEQ ID NO 5),
pro-IEPD-GrB--H6 C228T (SEQ ID NO 6), pro-IEPD-GrB--H6 C228V (SEQ
ID NO 7), and pro-IEPD-GrB--H6 C228F (SEQ ID NO 8).
29. A fusion protein according to claim 25, wherein the enzyme
Granzyme B is a human Granzyme B protease variant wherein the
Cystein residue no. 228 (chymotrypsinogen numbering) is mutated to
Phenylalanine.
30. A fusion protein according to claim 25, wherein the human
Granzyme B protease variant is as shown in SEQ ID NO 57.
31. A fusion protein according to claim 18, wherein the fusion
partner is an affinity-tag.
32. A fusion protein according to claim 31, wherein the
affinity-tag is selected from the group consisting of a
polyhistidine-tag, a polyarginine-tag, a FLAG-tag, a Strep-tag, a
c-myc-tag, a S-tag, a calmodulin-binding peptide, a
cellulose-binding peptide, a chitin-binding domain, a glutathione
S-transferase-tag, and a maltose binding protein.
33. A human Granzyme B protease variant wherein the Cystein residue
no. 228 (chymotrypsinogen numbering) is mutated to
Phenylalanine.
34. A human Granzyme B protease variant according to claim 33, as
shown in SEQ ID NO 57.
35. A method of cleaving a fusion protein comprising contacting
said fusion protein with the human Granzyme B protease variant
according to claim 33.
36. An isolated nucleic acid sequence encoding the fusion protein
according to claim 19 or the human Granzyme B protease variant
according to claim 33.
37. A recombinant vector comprising the isolated nucleic acid
sequence according to claim 36.
38. A host cell transformed with a vector according to claim
37.
39. A method for the production of a fusion protein according to
claim 18 or a human Granzyme B protease variant according to claim
33, comprising the steps of: (i) providing a recombinant vector
comprising the isolated nucleic acid sequence according to claim 36
operatively linked to a promotor, (ii) transforming a host cell
with said recombinant vector, (iii) culturing said host cell under
conditions to express said fusion protein or human Granzyme B
protease variant, and (iv) optionally isolating said fusion protein
or human Granzyme B protease variant.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for the
preparation of a polypeptide of interest in authentic form by
enzymatic cleavage of recombinantly produced fusion proteins by the
use of Granzyme B protease. Furthermore, the invention pertains to
fusion proteins comprising a Granzyme B cleavage site and to a
human Granzyme B variant.
BACKGROUND OF THE INVENTION AND PRIOR ART
[0002] The production and purification of recombinant polypeptides
such as pharmaceutical proteins in a highly purified and
well-characterized form, has become a major task within the area of
protein engineering in general, and in the pharmaceutical industry
in particular.
[0003] The preparation of such recombinant polypeptides relies
frequently on techniques which involve the production of the
polypeptides as fusion proteins or hybrid proteins, wherein a
protein or polypeptide of interest is fused to a carrier or a
fusion partner such as a polypeptide or a protein.
[0004] The presence of a fusion partner or carrier which is fused
to the polypeptide of interest has the advantages that it may
render the fusion protein more resistant to proteolytic
degradation, may facilitate enhanced expression and secretion,
improve solubility and allow for subsequent affinity purification
of the fusion protein. Also by fusion protein expression,
potentially bio-hazardous materials, such as peptide hormones, may
be produced in an inactive form which can then be activated
subsequently in vitro by cleaving off the fusion partner.
[0005] However, such fusion proteins themselves are not normally
suitable as end products as the fusion partner e.g. may affect the
biological activity or stability of the polypeptide of interest
and, if the protein is to be used clinically, may cause
antigenicity problems. Therefore it is necessary to cleave the
fusion protein to release the polypeptide of interest.
[0006] In principle this can be achieved by chemical or biochemical
methods such as enzymatic cleavage. However, it is important that
the cleavage is highly specific and only takes place in a cleavage
sequence between the polypeptide of interest and the fusion
partner, i.e. the junction region, but preferably not within the
polypeptide of interest itself, as this may e.g. severely affect
the bioactivity of the polypeptide of interest. Such methods employ
agents that act by hydrolysis of peptide bonds and the specificity
of the cleavage agent is determined by the identity of the amino
acid residue at or near the peptide bond which is cleaved.
[0007] Biochemical methods for cleavage of fusion proteins are
based on the use of proteases (proteolytic enzymes). However,
enzymatic cleavage of fusion proteins is limited in that the amino
acid(s) which are specific for the cleavage site can at the same
time also occur in the polypeptide of interest itself. Therefore,
enzymes are particularly suitable which, in order to cleave, not
only recognises one amino acid but rather a sequence of amino
acids, since the probability that a particular amino acid sequence
is present once again in the polypeptide of interest in addition to
the cleavage site between the polypeptide of interest and the
fusion partner is less the larger the number of amino acids
necessary for the recognition and cleavage of the cleavage
sequence.
[0008] Up till now, a number of proteases have been used for
enzymatic cleavage of fusion proteins by contacting the fusion
protein with a protease under appropriate conditions.
[0009] WO 03/010204 relates to a process for separating a
polypeptide of interest from a fusion protein by the use of
ubiquitin cleavage enzyme, which according to this document is an
enzyme that cleaves a peptide bond next to the amino acid sequence
RGG at the C-terminus of proteins such as ubiquitin.
[0010] U.S. Pat. No. 6,010,883 disclose a method wherein blood
clotting factor Xa (EC 3.4.21.6; a S1 serine-type peptidase formed
from the proenzyme factor X by limited proteolysis) is used for
cleaving off a fusion partner from a fusion protein. This protease
specifically cleaves after the amino acid sequence X--Y-Gly-Arg,
wherein X is Ile, Leu, Pro or Ala, and Y is Glu, Asp, Gln or Asn.
Factor Xa preferably cuts after the cleavage sequence
Ile-Glu-Gly-Arg.
[0011] Other prior art enzymes which have been suggested and used
in methods for specific cleavage of fusion proteins include tobacco
etch virus Nla proteinase, collagenase, enterokinase, subtilisin
and thrombin.
[0012] However, several problems may be encountered when using
proteolytic cleavage in fusion protein systems. One major problem
is the occurrence of non-specific proteolytic attack of the fusion
protein which results in cleavage at several locations and
consequently product loss and generation of contaminating
fragments. Also problems with inefficient or incomplete cleavage of
the fusion protein frequently occur with the presently known
enzymes. Such inefficient cleavage reduces the yield and may also
introduce heterogeneity to the purified protein resulting in the
recovery of only a small fraction of the desired protein.
[0013] A further problem that is associated with several of the
presently applied enzymes for fusion protein cleavage is that
spurious or extraneous amino acids are frequently attached to the
cleaved polypeptide product (the polypeptide of interest). These
amino acids are typically present when a linker is cleaved, and the
unrelated amino acid residues may have an effect on the properties
of the resulting polypeptide of interest. This may be critical for
proteins produced for human therapeutics. Therefore, it is highly
desirable to be able to produce pure authentic polypeptides free of
extraneous amino acid short sequences or residues.
[0014] The problem with extraneous amino acids remaining on the
polypeptide of interest after cleavage is illustrated in U.S. Pat.
No. 4,543,329 which describes a process for selectively cleaving a
fusion protein by the use of collagenase. However, the use of this
enzyme produces a polypeptide of interest with the extraneous amino
acid sequence Gly-Pro at its N-terminal. In order to obtain the
polypeptide of interest in authentic form, these extraneous amino
acids (Gly and Pro) must subsequently be removed in a further
reaction step by the use of one or more different amino peptidases
(such as aminoacylproline amino peptidase and proline amino
peptidase).
[0015] The problem is also illustrated in U.S. Pat. No. 5,427,927
which describes a process for sequence specific cleavage of fusion
proteins by the use of IgA protease, wherein a IgA protease
recognition site is inserted in the junction region of a fusion
protein which is subsequently cleaved with IgA protease. The
recognition site for the IgA protease is the amino acid sequence
Y-Pro.dwnarw.X-Pro, in which X can be any amino acid, Y can be one
or several arbitrary amino acids, and .dwnarw. denotes the cleavage
site. However, the proteins of interest which are formed after
cleavage by IgA protease, are characterised by having an X-Pro
extraneous amino sequence at its N-terminal, i.e. the resulting
polypeptide of interest is not in its native or authentic form.
[0016] Presently, the most widely used proteolytic enzymes for
fusion protein cleavage are the serine proteases factor Xa and
thrombin. However, both enzymes are known to perform non-specific
cleavage of fusion proteins. In addition, factor Xa has to be
isolated from bovine serum and as a consequence when it is used to
cleave proteins for therapeutic applications an extensive
purification and analysis is necessary afterwards in order to
detect pathogenic factors such as viruses and prions which may be
present (e.g. prions causing bovine spongiform encephalopathy).
Furthermore, these enzymes are rather expensive.
[0017] In view of these prior art shortcomings and drawbacks, it is
therefore an object of the present invention to provide an improved
method for enzymatic cleavage of fusion proteins.
[0018] It has been found by the present inventors, that the above
technical problems may be overcome by using Granzyme B protease (EC
3.4.21.79) for enzymatic cleavage of fusion proteins. Thus, it has
surprisingly been found that Granzyme B protease allows for highly
efficient cleavage of fusion proteins having a Granzyme B protease
cleavage site with a high degree of cleavage specificity. In
particular Granzyme B protease has proven to perform more specific
fusion protein cleavage than the presently and widely used protease
factor Xa. It has furthermore been found that Granzyme B cleavage
of fusion proteins that contain a Granzyme B recognition sequence
positioned between an N-terminal fusion partner and a C-terminal
polypeptide of interest, wherein the cleavage site is adjacent to
the polypeptide of interest, results in a polypeptide of interest
that have no extraneous amino acids derived from the cleavage site,
i.e. a polypeptide in authentic form. Thus, recombinant proteins of
interest with native amino acid sequence may be produced as a
result of fusion protein cleavage by Granzyme B. Also, the Granzyme
B protease has the advantage that it can be produced recombinantly.
Finally, it has been found that by substituting the Cysteine amino
acid no. 228 (chymotrypsinogen numbering) with Phenylalanine in
human Granzyme B, a higher final protein recovery can be obtained
when producing recombinant human Granzyme B.
SUMMARY OF THE INVENTION
[0019] Accordingly, the present invention relates in a first aspect
to a method for the preparation of a polypeptide of interest in
authentic form. The method comprises the steps of: (i) providing a
fusion protein comprising, from its N-terminal to its C-terminal
(a) a fusion partner, (b) a Granzyme B protease recognition site
comprising a Granzyme B protease cleavage site, (c) a polypeptide
of interest, wherein said cleavage site being adjacent to the
polypeptide of interest, and (ii) contacting the fusion protein
with Granzyme B protease (EC 3.4.21.79) to cleave it at the
cleavage site to yield the polypeptide of interest in authentic
form.
[0020] In a further aspect there is provided a fusion protein
comprising, from its N-terminal to its C-terminal, (a) a fusion
partner, (b) a Granzyme B protease recognition site comprising a
Granzyme B protease cleavage site, and (c) a polypeptide of
interest, wherein said cleavage site being adjacent to the
polypeptide of interest.
[0021] There is also provided a human Granzyme B protease variant
wherein the Cystein residue no. 228 (chymotrypsinogen numbering) is
mutated to Phenylalanine.
[0022] In still further aspects there is provided an isolated
nucleic acid sequence encoding such a fusion protein or human
Granzyme B protease variant, a recombinant vector comprising the
isolated nucleic acid sequence, a host cell transformed with such a
vector, and a method for the production of the fusion protein or
the human Granzyme B protease variant which comprises the steps of
(i) providing such a recombinant vector which is operatively linked
to a promotor, (ii) transforming a host cell with the recombinant
vector, (iii) culturing the host cell under conditions to express
the fusion protein, and (iv) optionally isolating the fusion
protein or the human Granzyme B protease variant.
DETAILED DISCLOSURE OF THE INVENTION
[0023] In one aspect the present invention relates to a method for
preparing a polypeptide of interest in authentic form by enzymatic
cleavage of fusion proteins. Accordingly, the method comprises, as
is mentioned above, a step of providing a fusion protein comprising
from its N-terminal to its C-terminal, a fusion partner, a Granzyme
B protease recognition site comprising a Granzyme B protease
cleavage site and a polypeptide of interest, wherein the cleavage
site is placed adjacent to the polypeptide of interest. The fusion
protein is subsequently contacted with Granzyme B protease to
cleave the fusion protein at the Granzyme B protease cleavage site
to yield the polypeptide of interest in authentic form. The term
"fusion protein" as used herein, refers to a polypeptide which
comprises protein domains from at least two different proteins.
[0024] In accordance with the present invention there is provided a
method for producing polypeptides of interest in authentic form. As
used herein, the term "authentic form" refers to a polypeptide
which comprises the amino acid sequence thereof without any
additional amino acid residues. As described above, a major problem
associated with several of the presently applied enzymes for fusion
protein cleavage is that spurious or extraneous amino acids
frequently remains attached to the cleaved polypeptide product,
i.e. resulting in a polypeptide which is not in an "authentic
form". Thus, in the present context the polypeptide of interest in
authentic form refers to a polypeptide having the same primary
amino acid sequence as that encoded by the native gene sequence
coding for the polypeptide of interest, i.e. it does not contain
any non-native amino acids. The term "native gene sequence" is not
necessarily a gene sequence that occurs in nature, but it may also
be partly or completely artificial. Likewise it will be appreciated
that a polypeptide of interest in authentic form not necessarily is
a polypeptide that occurs in nature, but it may also be partially
or completely artificial. In contrast, a "non-authentic"
polypeptide contains at least one amino acid which is not encoded
for by the native gene sequence coding for the polypeptide of
interest.
[0025] In accordance with the invention, the junction region
between the polypeptide of interest and the fusion partner
comprises a Granzyme B protease recognition site which has a
Granzyme B protease cleavage site. Such a recognition site refers
to a defined amino acid sequence that allows Granzyme B to
recognize and to cleave the junction region between the fusion
partner and the polypeptide of interest. The cleavage site is thus
to be understood as the site between two amino acids of an amino
acid sequence at which the cleavage of the fusion protein takes
place. The junction region may be in the form of a linker sequence
of any suitable length which is not part of the polypeptide of
interest. However, in order to obtain the polypeptide of interest
in authentic form, the Granzyme B cleavage site is positioned
adjacent to the N-terminus of the polypeptide of interest in order
to allow for specific cleavage of the fusion protein at its
N-terminus without resulting in spurious or extraneous amino acids
remaining attached to the resulting polypeptide of interest. Hence,
the term "adjacent to" imply that the Granzyme B recognition
sequence, which in some embodiments may be preceded by or be a part
of a linker sequence, is positioned such that the Granzyme B
cleavage site is flanking the N-terminus of the polypeptide of
interest.
[0026] Granzymes are granule-stored serine proteases that are
implicated in T cell and natural killer cell-mediated cytotoxic
defence reactions after target cell recognition. The principal
function of granzymes is to induce the death of virus-infected and
other potentially harmful cells. Granzyme B is one type of
granzymes, and upon target cell contact it is directionally
exocytosed and enters target cells assisted by perforin (a
cytolytic protein expressed by cytotoxic T cells and natural killer
cells). Granzyme B processes and activates various pro-caspases,
thereby inducing apoptosis in the target cell. In accordance with
the invention, the term "Granzyme B protease" (also referred to
herein as GrB) includes enzymes which are or may be classified
under the Enzyme Commission number EC 3.4.21.79 in Enzyme
nomenclature database, Release 34, February 2004
(http://www.expasy.org/enzyme). Thus, in accordance with the
invention any suitable Granzyme B protease may be used including
human Granzyme B protease, mouse Granzyme B protease and rat
Granzyme B protease. It is generally preferred to use human
Granzyme B, when the method in accordance with invention is used
for the preparation of human therapeutic protein products. Human
Granzyme B protease occurs in most human tissues where its
biological function is well known. Therefore, the presence of trace
amounts of residual Granzyme B protease in the final therapeutic
protein product exhibit a minimal risk for the patient to whom the
therapeutic protein product is administered. Thus, it is known that
if active Granzyme B protease is injected into the human blood
stream it is swiftly trapped by alpha-2-macroglobulin and the
complex is cleared via the LRP scavenging receptor. Granzyme B
protease is also known under the alternative name "Cytotoxic
t-lymphocyte proteinase 2".
[0027] Granzyme B protease is known to have a preference for
cleaving after aspartate residues (D), and Granzyme B is the only
mammalian serine protease known to have this P1-proteolytic
specificity. Hence, in accordance with the invention it is
contemplated that the Granzyme B cleavage site in useful
embodiments at least comprises an aspartate residue at the P1
position located N-terminally to the cleavage site. Some of the
presently known Granzyme B protease recognition sites are disclosed
in Harris et al (1998). Thus, in useful embodiments, the
recognition site has an amino acid sequence of the general formula:
P4 P3 P2 P1.dwnarw. located N-terminally to the cleavage site,
wherein P4 preferably is amino acid I or V, P3 preferably is amino
acid E, Q or M, P2 is X, where X denotes any amino acid, P1
preferably is amino acid D, and .dwnarw. is the cleavage site for
the Granzyme B protease.
[0028] It was found by the present inventors, that Granzyme B
protease is capable of cleaving off polypeptides of interest from a
fusion protein, without leaving any non-native amino acids on the
polypeptide of interest. In particular it was surprisingly found
that Granzyme B would recognise and cleave off polypeptides from a
fusion protein after the P1 position without any strict
requirements for specific amino acid residues at the P1'-P4'
positions, i.e. the amino acid positions following the cleavage
site. This is contrary to the findings in the prior art. In e.g.
Sun et al. (2001) it is concluded that the P1'-P4' residues of
Granzyme B substrates are important for substrate binding, and that
highest affinity for the substrate is observed when an acidic P4'
residue is present (i.e. either aspartate or glutamic acid).
Furthermore, Harris et al. (1998) concluded that Granzyme B has a
strong preference for glycine residue at the P2' position. Despite
these prior art findings, it has now been established that Granzyme
B protease may be generally used for cleaving off polypeptides of
interest from fusion proteins, without the need for specific amino
acid residues at the P1'-P4' positions.
[0029] As mentioned above, a further particular advantage of the
present invention is the finding that Granzyme B protease allows
for highly efficient cleavage of fusion proteins having a Granzyme
B protease cleavage site positioned N-terminally to the polypeptide
of interest with a high degree of cleavage specificity. In
particular it has been found that Granzyme B protease perform more
specific fusion protein cleavage than the presently and widely used
protease for fusion protein cleavage, namely factor Xa. Thus, as
will be apparent from the below examples, it was found that when
five different fusion proteins previously made as factor Xa
cleavable fusion proteins, were cleaved by Granzyme B protease, it
resulted in a cleavage performance that was as specific as or even
more specific than that found with factor Xa. This may e.g. be seen
from Example 8 and the accompanying FIG. 11 illustrating an
extended time course PAGE analysis of the digestion of the fusion
proteins H6-IEGR--RAP and H6-IEPD-RAP with factor Xa and Granzyme
B, respectively. It is clearly seen from this experiment that after
30 min. there was an essentially complete cleavage of the fusion
proteins with both proteases. However, more breakdown products were
produced by the use of factor Xa as compared to Granzyme B. This
clearly shows that Granzyme B is highly specific, and even more
specific than the widely used protease factor Xa. The high
versatility and great flexibility of Granzyme B is further
substantiated herein by the findings that Granzyme B is both
capable of cleaving off relatively short N-terminal tags such as a
hexa-His tail from a polypeptide of interest, and to cleave between
a fusion partner and a polypeptide of interest which are very
closely connected by a short linker sequence.
[0030] Although not necessary, it may in certain embodiments be
advantageous to select the polypeptide of interest such that the
polypeptide of interest, when it is a part of the fusion protein,
N-terminally comprises the amino acids P1' and P2' resulting in the
general Granzyme B recognition site formula P4 P3 P2
P1.dwnarw.P1'P2' wherein P1' is X, where X denotes any amino, and
P2' is G. Although Granzyme B protease has no strict amino acid
selectivity for the P1' position, there is a general preference for
large hydrophobic amino acids at this position including Trp (T),
Leu (L), Phe (F) and lie (I). Thus, in one useful embodiment the
amino acid at position P1' is selected from T, L, F and I. It may
in certain embodiments by advantageous not to include Pro (P) at
the P2' position. It may in a further aspect of the invention be
advantageous that the polypeptide of interest is selected such that
it, when being part of the fusion protein, N-- terminally comprises
an acidic amino acid at the P4' position, such as D or E.
[0031] In the present context the terms "amino acid" and "amino
acid residues" refer to all naturally occurring L-alpha-amino
acids. This definition is meant to include norleucine, ornithine,
and homocysteine. The amino acids are identified by either the
three-letter or single-letter designations:
[0032] Asp, D: aspartic acid Ile, I: isoleucine
[0033] Thr, T: threonine Leu, L: leucine
[0034] Ser, S: serine Tyr, Y: tyrosine
[0035] Glu, E: glutamic acid Phe, F: phenylalanine
[0036] Pro, P: proline His, H: histidine
[0037] Gly, G: glycine Lys, K: lysine
[0038] Ala, A: alanine Arg, R: arginine
[0039] Cys, C: cysteine Trp, W: tryptophan
[0040] Val, V: valine Gln, Q: glutamine
[0041] Met, M: methionine Asn, N: asparagine
[0042] Nle, J: norleucine Orn, O: ornithine
[0043] Hcy, U: homocysteine Xxx, X: any L-alpha-amino acid.
[0044] In further useful embodiments, the Granzyme B protease
recognition site has an amino acid sequence which is selected from
ICPD.dwnarw., IEAD.dwnarw., IEPD.dwnarw., IETD.dwnarw.,
IQAD.dwnarw., ISAD.dwnarw., ISSD.dwnarw., ITPD.dwnarw.,
VAPD.dwnarw., VATD.dwnarw., VCTD.dwnarw., VDPD.dwnarw.,
VDSD.dwnarw., VEKD.dwnarw., VEQD.dwnarw., VGPD.dwnarw.,
VEID.dwnarw., VRPD.dwnarw., VTPD.dwnarw., LEED.dwnarw.,
LEID.dwnarw., LGND.dwnarw., LGPD.dwnarw., AQPD.dwnarw., where
.dwnarw. is the cleavage site for Granzyme B. These recognition and
cleavage sites have previously been described by Casciola-Rosen et
al. (1999).
[0045] In accordance with the invention the terms "polypeptide of
interest" or "desired polypeptide" refer to the polypeptide whose
expression is desired within the fusion protein. As used herein,
the term "polypeptide" should not necessarily indicate a limit on
the size of the desired polypeptide of interest. Thus, this term is
to be interpreted in its broadest sense, and hence includes
peptides on the order of up to 50 or more amino acids, including
oligopeptides such as di-, tri, tetra-, penta-, and hexa-peptides,
polypeptides and proteins. The polypeptide of interest may by an
intermediate product or a final product which can for example be
used in the field of medicine, in research, in environmental
protection, or in industrial processes or products. As previously
described, in the fusion protein the polypeptide of interest is
joined or fused with another protein or protein domain, the fusion
partner, to allow for e.g. enhanced stability of the polypeptide of
interest and ease of purification of the fusion protein. In useful
embodiments the polypeptide of interest is a protein such as a
secreted protein. Secreted proteins have various industrial
applications, including as pharmaceuticals, and diagnostics. Most
protein drugs available at present, such as thrombolytic agents,
interferons, interleukins, erythropoietins, colony stimulating
factors, and various other cytokines, are secreted proteins. In
useful embodiments the polypeptide of interest is a polypeptide
hormone such as somatotrophin, glucagon, insulin or interferon, a
single chain antibody variable region fragment (scfv), or an
apolipoprotein such as apolipoprotein a-i (apoA-I), apolipoprotein
A-II, or apolipoprotein A-IV.
[0046] In a further aspect of the invention the polypeptide of
interest is an enzyme, such as Granzyme B. Thus, by providing a
fusion protein in accordance with the invention and selecting
Granzyme B protease as the polypeptide of interest, there is
provided a self-activating Granzyme B protease which offers the
possibility of providing inactive pro-Granzyme B which subsequently
may be activated, in principle, by the addition of a single
molecule of active Granzyme B protease. Thereby, there is provided
pro-Granzyme B which is not dependent on the addition of e.g.
external activator biologicals for its activation. As will be
apparent from the following examples, the Granzyme B self
activation was found to progress quantitatively to completion, and
self activating samples of Granzyme B protease subjected to further
incubation for several days were found to retain stable activity
levels and produce minimal amounts of autolysis products. This
clearly demonstrates that self-activating Granzyme B protease has
the advantage of being highly stable to autolysis (cannibalism), as
shown in Example 5 and FIG. 3.
[0047] The fusion partner may, in accordance with the invention, be
of any suitable kind provided that it is a peptide, oligopeptide,
polypeptide or protein, including a tetra-peptide, penta-peptide
and a hexa-peptide. It may be selected such that it renders the
fusion protein more resistant to proteolytic degradation,
facilitate enhanced expression and secretion of the fusion protein,
improve solubility, and allow for subsequent affinity purification
of the fusion protein.
[0048] The fusion protein of the present invention may in useful
embodiments comprise a fusion partner which is an affinity-tag.
Such an affinity-tag may e.g. be an affinity domain which permits
the purification of the fusion protein on an affinity resin. The
affinity-tag may also be a polyhistidine-tag including hexahis-tag,
a polyarginine-tag, a FLAG-tag, a Strep-tag, a c-myc-tag, a S-tag,
a calmodulin-binding peptide, a cellulose-binding peptide, a
chitin-binding domain, a glutathione S-transferase-tag, or a
maltose binding protein.
[0049] As mentioned above, any suitable Granzyme B protease may be
used in accordance with the invention including human Granzyme B
protease, mouse Granzyme B protease and rat Granzyme B protease.
However, as will be apparent from the following Examples, it was
found that by substituting the Cystein residue no. 228
(chymotrypsinogen numbering) with Phenylalanine in human Granzyme B
protease, there is provided a human Granzyme B protease variant
which results in a higher final protein yield when producing human
Granzyme B, even without affecting the cleavage specificity or
activity of the resulting Granzyme B to any noticeable degree. This
finding is contrary to what would have been expected, as the amino
acid Phenylalanine (aromatic amino acid) is chemically very
different from Cysteine (hydrophilic amino acid) and would
therefore not normally be the choice for a Cysteine substitution.
Thus, in a presently preferred embodiment the Granzyme B protease
according to the invention is a human Granzyme B protease variant
wherein the Cystein residue no. 228 (chymotrypsinogen numbering) is
mutated to Phenylalanine. It will be appreciated that the term
"human Granzyme B protease variant" also includes variants which in
addition to the Cystein residue no. 228 (chymotrypsinogen
numbering) mutation, have further variations in the full-length
sequence of native human Granzyme B protease or in various domains,
e.g. Granzyme B protease variants wherein one or more amino acid
residues are added, or deleted, at the N-- or C-terminus of the
full-length native Granzyme B amino acid sequence. Such further
variations can be made, for example, using any of the techniques
and guidelines for conservative and non-conservative mutations
known in the art. Variations may be a substitution, deletion or
insertion of one or more codons encoding the human Granzyme B
protease that results in a change in the amino acid sequence of the
human Granzyme B protease as compared with the native sequence of
human Granzyme B protease preferably without adversely affecting
the human Granzyme B protease specificity and/or activity. Also
fragments of the full-length native Granzyme B amino acid sequence,
such as the activated form, having a Cystein residue no. 228
mutation is included in the meaning of "human Granzyme B protease
variant". In a useful embodiment the Granzyme B protease variant is
the variant shown in SEQ ID NO 57.
[0050] In general the fusion partner will typically be selected on
the basis of characteristics contributing to ease isolation, most
desirable being those that are readily secreted by the
microorganisms producing the fusion protein. Polyhistidine
sequences, glutathione S-transferase and maltose binding protein,
for example, are generally preferred as there are readily available
affinity columns to which they can be bound and eluted from.
[0051] The method according to the invention may in useful
embodiments include a subsequent isolation step for isolating the
polypeptide of interest which is formed by the enzymatic cleavage
of the fusion protein. This isolation step can be performed by any
suitable means known in the art for protein isolation, including
the use of ion exchange, fractionation by size and affinity
purification, the choice of which depending on the character of the
polypeptide of interest. Thus the polypeptide of interest may for
the purpose of affinity purification e.g. further comprise a
C-terminally linked affinity-tag in to order to provide for
isolation of the resulting polypeptide of interest using e.g. the
above mentioned affinity-tag systems.
[0052] In accordance with the invention the fusion protein is
contacted with Granzyme B protease to cleave the fusion protein at
the Granzyme B protease cleavage site adjacent to the polypeptide
of interest to yield the polypeptide of interest in authentic form.
This reaction may be carried out batchwise using free Granzyme B,
or it may be carried out by using Granzyme B protease in an
immobilised form, e.g. via adsorption, covalent binding, entrapment
or membrane confinement. Suitable carriers for immobilised Granzyme
B protease include conventional carriers such as polyacrylamide,
chitin, dextran, kappa carrageenan, celite and cellulose.
Immobilisation of enzymes by their covalent coupling to insoluble
matrices is an extensively used technique. Lysine residues are
found to be the most generally useful groups for covalent bonding
of enzymes to insoluble supports due to their widespread surface
exposure and high reactivity. Thus, in useful embodiments the
Granzyme B protease is immobilised via a lysine amino acid residue.
In a further aspect the Granzyme B protease is immobilised via its
C-terminus, e.g. by means of a polyhistidine-tag, including a
hexa-histidine-tag. The reaction may also be conducted by using a
free Granzyme B protease in combination with a membrane-type
bioreactor, or using a continuous type bioreactor together with an
immobilised Granzyme B protease.
[0053] As will be apparent from the following examples, it has
surprisingly been found that the time required for the cleavage of
free fusion proteins (i.e. not immobilised) which comprises a
polyhistidine fusion partner such as hexa-histidine, may be
decreased dramatically if the fusion protein is contacted with
Granzyme B protease in the presence of Ni.sup.2+ ions and
Nitrilotriacetic Acid (NTA). It is contemplated that the main
reason for this remarkable increase in cleavage speed, is that the
Ni.sup.2+ ions bind the N-- terminal polyhistidine fusion partner
of the fusion protein and facilitate access for the Granzyme B
protease to the cleavage site. Additionally it is also considered
that the addition of NTA will shield the Ni.sup.2+ ions in solution
in a similar fashion as on a Ni.sup.2+--NTA agarose column, and
thereby avoid precipitation of both the fusion protein and the
resulting protein. When performing such a cleavage process, it is
generally preferred that the concentration of Ni.sup.2+ is in the
range of 1-20 mM, and the concentration of NTA is in the range of
1-20 mM. Furthermore, the temperature is preferably in the range of
15-50.degree. C., including the range of 20-45.degree. C. In a
preferred embodiment, the temperature is in the range of
20-30.degree. C., such as about 23.degree. C. In another slightly
less preferred embodiment, the temperature is in the range of
30-45.degree. C., such as about 37.degree. C. The optimal
temperature range, though, must be determined for each fusion
protein, since it depends in part on the stability of the fusion
protein at the different temperatures.
[0054] In accordance with the invention there is as already
described above, also provided a fusion protein comprising, from
its N-terminal to its C-terminal, (a) a fusion partner, (b) a
Granzyme B protease recognition site comprising a Granzyme B
protease cleavage site, and (c) a polypeptide of interest, wherein
the cleavage site being adjacent to the polypeptide of interest. In
useful embodiments, the polypeptide of interest is Granzyme B,
which thereby provides for a self-activating Granzyme B protease.
In particular there is provided self-activating human pro-Granzyme
B fusion proteins comprising from the N-terminal to the C-terminal,
a seven amino acid residue pro-sequence having a Granzyme B
recognition site and cleavage site followed by the amino acid
sequence for activated human Granzyme B, and finally a
hexa-Histidine tag (H6) fused to the C-- terminal of the Granzyme
B. Thus, the Granzyme B cleavage site is located adjacent to the
Ile16 (chymotrysinogen numbering) of the amino acid sequence for
activated Granzyme B. More particular there is provided the human
self-activating Granzyme B fusion proteins pro-IEPD-GrB--H6 (SEQ ID
NO 2) and pro-IEAD-GrB--H6 (SEQ ID NO 3). As also previously
described, it was found to be advantageous, to substitute the
Cystein residue no. 228 (chymotrypsinogen numbering) with alanine
(A), threonine (T), valine (V), or phenylalanine (F) by
site-directed mutation. Thus, in further useful embodiments there
is provided self-activating fusion proteins selected from the group
consisting of pro-IEPD-GrB--H6 C228A (SEQ ID NO 5),
pro-IEPD-GrB--H6 C228T (SEQ ID NO 6), pro-IEPD-GrB--H6 C228V (SEQ
ID NO 7), and pro-IEPD-GrB--H6 C228F (SEQ ID NO 8).
[0055] The fusion protein or the Granzyme B protease variant of the
present invention may be expressed in any suitable standard protein
expression system by culturing a host transformed with a vector
encoding the fusion protein under such conditions that the fusion
protein is expressed. Preferably, the expression system is a system
from which the desired fusion protein may readily be isolated and
refolded in vitro. As a general matter, prokaryotic expression
systems are preferred since high yields of protein can be obtained
and efficient purification and refolding strategies are available.
However, numerous host cells may be selected as appropriate for
transformation and expression of the described fusion protein,
including mammalian insect, fungal and bacterial host cells which
are particularly desirable. Commonly used bacterial strains include
Bacillus and Escherichia, including E. coli. Thus, it is well
within the abilities and discretion of the skilled artisan, without
undue experimentation, to choose an appropriate or favourite host
and expression system. Similarly, once the primary amino acid
sequence for the fusion protein of the present invention is chosen,
one of ordinary skill in the art can easily design appropriate
recombinant nucleic acid sequence or DNA constructs encoding the
fusion proteins or the Granzyme B protease variant of the
invention, taking into consideration such factors as codon biases
in the chosen host, the need for secretion signal sequences in the
host, the introduction of proteinase cleavage sites within the
signal sequence, and the like. These recombinant DNA constructs may
be inserted in-frame into any of a number of expression vectors
appropriate to the chosen host. The choice of an appropriate or
favourite expression vector is, again, a matter well within the
ability and discretion of the skilled practitioner. Preferably, the
expression vector will include a strong promoter to drive
expression of the recombinant constructs.
[0056] Finally, there is provided a method for the production of a
fusion protein or the Granzyme B protease variant according to
invention which comprises the steps of (i) providing a recombinant
vector comprising the isolated nucleic acid sequence encoding the
fusion protein or the Granzyme B protease variant of the invention
which is operatively linked to a promotor, (ii) transforming a host
cell with this recombinant vector, (iii) culturing the host cell
under conditions to express the fusion protein, and (iv) optionally
isolating the fusion protein or the Granzyme B protease
variant.
[0057] The invention will now be described by way of illustration
in the following non-limiting examples and figures.
DESCRIPTION OF THE FIGURES
[0058] FIG. 1 shows the activity of an incubation of GrB--H6 with
FX.sub.a followed for several days using the following colorimetric
assay: 500 .mu.l buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.0), 4
.mu.l 100 mM Ac--IEPD-pNA and 5 .mu.l GrB--H6. A mixture of 100
.mu.l GrB--H6 (approximately 10 .mu.g) with 1 .mu.l FX.sub.a (1
mg/ml) was kept at 4.degree. C. during the incubation, and the
activity was measured after 0 hours, 2 hours, 5 hours, 19 hours, 2
days and 5 days.
[0059] FIG. 2 shows the activity of both GrB--H6 and GrB--H6 C228F
towards several chromogenic substrates: Ac--IEPD-pNA, Ac-LEED-pNA,
Ac--VEID-pNA, Ac--YVAD-pNA, and Ac-DEVD-pNA. The activity assay was
carried out in 500 .mu.l 100 mM HEPES pH 7.75 with a substrate
concentration of 400 .mu.M and 1 .mu.g protease added for each
measurement. All measurements were done at 23.degree. C. and in
triplicate, and the activities obtained were normalized by setting
the activity measured on Ac--IEPD-pNA to 100%.
[0060] FIG. 3. panel (A) shows the SDS PAGE of samples from the
incubation of GrB--H6 C228F in 100 mM HEPES pH 7.4 at 4.degree. C.,
23.degree. C., and 37.degree. C.
[0061] Description of lanes A-N:
[0062] A: Molecular weight marker
[0063] B: GrB--H6 C228F, before incubation
[0064] C: GrB--H6 C228F incubated at 4.degree. C. for 1 day
[0065] D: GrB--H6 C228F incubated at 4.degree. C. for 3 days
[0066] E: GrB--H6 C228F incubated at 4.degree. C. for 6 days
[0067] F: GrB--H6 C228F incubated at 4.degree. C. for 15 days
[0068] G: GrB--H6 C228F incubated at 23.degree. C. for 1 day
[0069] H: GrB--H6 C228F incubated at 23.degree. C. for 3 days
[0070] I: GrB--H6 C228F incubated at 23.degree. C. for 6 days
[0071] J: GrB--H6 C228F incubated at 23.degree. C. for 15 days
[0072] K: GrB--H6 C228F incubated at 37.degree. C. for 1 day
[0073] L: GrB--H6 C228F incubated at 37.degree. C. for 3 days
[0074] M: GrB--H6 C228F incubated at 37.degree. C. for 6 days
[0075] N: GrB--H6 C228F incubated at 37.degree. C. for 15 days
[0076] Lane B shows the intact GrB--H6 C228F. This band of intact
protease can be seen in all lanes (1). In lanes I-N another band
appears (2), which must be a degradation product of the protease,
possibly arisen by auto cleavage between Asp50 and Phe51 in the
sequence IQDD.dwnarw.FV.
[0077] FIG. 3, Panel (B) shows the activity of the samples of
GrB--H6 C228F incubated in 100 mM HEPES pH 7.4 at 4.degree. C.,
23.degree. C., and 37.degree. C. measured after 0, 6, (10), and 15
days. The activity was measured in 500 .mu.l 100 mM HEPES pH 7.4
and 400 .mu.M Ac--IEPD-pNA with 0.2 pg GrB--H6 C228F from the
incubations added for each measurement.
[0078] FIG. 4 shows the SDS PAGE of samples from the H6-TripUB
IEPD.dwnarw.SP and H6-IEPD-TN123 incubations after 12 hours
incubation with GrB--H6.
[0079] Description of the lanes A-J:
[0080] A: Molecular weight marker
[0081] B: H6-TripUB IEPD.dwnarw.SP alone after 12 hours
incubation
[0082] C: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+1 .mu.l GrB--H6 after
12 hours incubation
[0083] D: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+10 .mu.l GrB--H6 after
12 hours incubation
[0084] E: H6-FX-TripUB incubated with FX.sub.a
[0085] F: H6-IEPD-TN123 alone after 12 hours incubation
[0086] G: 200 .mu.l H6-IEPD-TN123+1 .mu.l GrB--H6 after 12 hours
incubation
[0087] H: 200 .mu.l H6-IEPD-TN123+10 .mu.l GrB--H6 after 12 hours
incubation
[0088] I: GrB--H6 alone in the same concentration as in lanes D and
H
[0089] J: Murine H6-FX-TN123 cleaved with FX.sub.a
[0090] Lane B shows non-cleaved H6-TripUB IEPD.dwnarw.SP (1), where
no GrB--H6 was added, while lanes C and D show the two incubations
with 1 and 10 .mu.l GrB--H6 added. In both these lanes the product
of the cleavage reaction; correctly cleaved H6-TripUB
IEPD.dwnarw.SP (2) is seen in addition to the non-cleaved fusion
protein. In lane E the construct H6-FX-TripUB, containing the
FX.sub.a recognition site IQGR in place of the GrB recognition site
IEPD, is cleaved by FX.sub.a giving a product of the same size as
the GrB--H6 cleaved H6-TripUB IEPD.dwnarw.SP.
[0091] Lanes F-J show the GrB--H6+H6-IEPD-TN123 incubations after
12 hours. In lane F is shown non-cleaved H6-IEPD-TN123 (3). Lanes G
and H show how H6-IEPD-TN123 is cleaved by GrB--H6 when no
Ca.sup.2+ is present (4). The band pattern is explained in FIG. 12.
In lane J the murine H6-FX-TN123 construct has been cleaved by
FX.sub.a showing the size of the correctly cleaved product.
[0092] Marked by (5) in the figure is the position of GrB--H6 with
the same concentration as in the samples with 10 .mu.l GrB--H6
added.
[0093] FIG. 5 shows the SDS PAGE of the samples from the
GrB--H6+H6-TripUB IEPD.dwnarw.SP incubations after 12, 19 and 24
hours of incubation, as well as the samples from the
GrB--H6+H6-IEPD-TN123 incubations.
[0094] Description of the lanes A-K:
[0095] A: Molecular weight marker
[0096] B: H6-TripUB IEPD.dwnarw.SP alone after 12 hours
incubation
[0097] C: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+1 .mu.l GrB--H6 after
12 hours incubation
[0098] D: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+1 .mu.l GrB--H6 after
19 hours incubation
[0099] E: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+1 .mu.l GrB--H6 after
24 hours incubation
[0100] F: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+10 .mu.l GrB--H6 after
19 hours incubation
[0101] G: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+10 .mu.l GrB--H6 after
24 hours incubation
[0102] H: GrB--H6 alone diluted as in F and G
[0103] I: H6-IEPD-TN123 alone after 12 hours incubation
[0104] J: 200 .mu.l H6-IEPD-TN123+1 .mu.l GrB--H6 after 12 hours
incubation
[0105] K: 200 .mu.l H6-IEPD-TN123+10 .mu.l GrB--H6 after 12 hours
incubation
[0106] Lane B shows non-cleaved H6-TripUB IEPD.dwnarw.SP (1). In
lanes C-E the correctly cleaved product appears in all lanes,
marked by (2) in the figure. The more GrB--H6 added and the longer
the incubation time, the more cleavage product appears in the
lanes.
[0107] Lanes I, J, and K in FIG. 3 are identical to lanes F, G, and
H in FIG. 2 with the H6-IEPD-TN123+GrB--H6 incubations, though a
larger sample has been run on the gel in FIG. 3. The bands are
therefore much clearer than in FIG. 2. The band marked with (3) is
non-cleaved H6-IEPD-TN123 and the band pattern marked with (4),
(5), (6) and (7) is explained in FIG. 12.
[0108] FIG. 6 explains the simple band pattern observed in FIGS. 2
and 3. When no GrB--H6 is added, no cleavage occurs and only the
band from the non-cleaved fusion protein is seen in the gel. When
GrB--H6 is added, the small N-terminal sequence is cleaved off and
the correctly cleaved product appears on the gel in addition to the
remaining non-cleaved fusion protein. The small N-terminal sequence
cleaved off by GrB--H6 is too small to be visualized on the SDS
gel.
[0109] FIGS. 7, 8 and 9 show the SDS PAGE of the samples from the
H6-TripUB IEPD.dwnarw.SP+GrB--H6 incubations at 23.degree. C. (FIG.
5), 37.degree. C. (FIG. 6) and 42.degree. C. (FIG. 7) with no
addition (1), addition of 4.2 mM Ni.sup.2+ (2) and addition of 4.2
mM Ni.sup.2++5 mM NTA (3). Description of the lanes A-K (same for
all temperatures):
[0110] A: Molecular weight marker
[0111] B: Non-cleaved H6-TripUB IEPD.dwnarw.SP
[0112] C: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6, no
addition
[0113] D: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6, no
addition
[0114] E: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6, no
addition
[0115] F: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6, 4.2
mM Ni.sup.2+
[0116] G: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6, 4.2
mM Ni.sup.2+
[0117] H: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6, 4.2
mM Ni.sup.2+
[0118] I: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6, 4.2
mM Ni.sup.2+ and 5 mM NTA
[0119] J: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6, 4.2
mM Ni.sup.2+ and 5 mM NTA
[0120] K: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6, 4.2
mM Ni.sup.2+ and 5 mM NTA
[0121] In lanes C-E (1) in all three figures where no Ni.sup.2+ or
NTA was added, the H6-TripUB IEPD.dwnarw.SP fusion protein is
cleaved at different rates for different temperatures. After 22
hours at 23.degree. C. approximately 40% of the fusion protein has
been cleaved. At 37.degree. C. and 42.degree. C. more was cleaved
after 22 hours than at 23.degree. C., approximately 60% at
37.degree. C. and 50% at 42.degree. C.
[0122] Because of the precipitation of protein observed at
37.degree. C. and 42.degree. C. with 4.2 mM Ni.sup.2+ no further
cleavage of the fusion protein after 2 hours incubation was seen in
the gel. Therefore less protein was seen in lanes F-H (2) in FIGS.
6 (37.degree. C.) and 7 (42.degree. C.) than in lanes F-H (2) in
FIG. 5 (23.degree. C.). In FIG. 5, lanes F-H (2), approximately 50%
of the fusion protein was cleaved to product after 22 hours
incubation, which is more than was cleaved with no addition of
Ni.sup.2+.
[0123] No precipitation was observed with 4.2 mM Ni.sup.2++5 mM NTA
added to the incubations. In FIG. 5 lanes I-K (3) more product is
seen than in lanes C-E (1) and F-H (2), so after 22 hours
incubation at 23.degree. C. with both Ni.sup.2+ and NTA present
approximately 60% of the fusion protein has been cleaved compared
to only about 40% with no addition and 50% with Ni.sup.2+
alone.
[0124] By further increasing the temperature to 37.degree. C. (FIG.
6 lanes I-K (3)) and 42.degree. C. (FIG. 7 lane I-K (3)) an even
greater increase in the rate of cleavage is seen. After 22 hours of
incubation at 37.degree. C. almost all the fusion protein is
cleaved to the correct product. A little less is cleaved at
42.degree. C. after 22 hours.
[0125] FIG. 10 shows the SDS PAGE of the samples from the
H6-IEPD-RAP incubations with 1 or 10 .mu.l GrB--H6. Description of
the lanes (A-L):
[0126] A: Molecular weight marker
[0127] B: H6-IEPD-RAP alone after 5 hours incubation
[0128] C: 200 .mu.l H6-IEPD-RAP+1 .mu.l GrB--H6 after 5 hours
incubation
[0129] D: 200 .mu.l H6-IEPD-RAP+10 .mu.l GrB--H6 after 5 hours
incubation
[0130] E: H6-IEPD-RAP alone after 23 hours incubation
[0131] F: 200 .mu.l H6-IEPD-RAP+1 .mu.l GrB--H6 after 23 hours
incubation
[0132] G: 200 .mu.l H6-IEPD-RAP+10 .mu.l GrB--H6 after 23 hours
incubation
[0133] H: H6-IEGR--RAP cut partly with FX.sub.a, purified
[0134] I: H6-IEGR--RAP cut almost completely with F), purified
[0135] J: H6-IEPD-RAP alone after 26 hours incubation
[0136] K: 200 .mu.l H6-IEPD-RAP+1 .mu.l GrB--H6 after 26 hours
incubation
[0137] L: 200 .mu.l H6-IEPD-RAP+10 .mu.l GrB--H6 after 26 hours
incubation
[0138] Non-cleaved H6-IEPD-RAP (1) is shown in lanes B, E, and J.
In lanes C and D it is clear that all the H6-IEPD-RAP has been
cleaved to give the final product (2) after only 5 hours incubation
with either 1 or 10 .mu.l GrB--H6 as described above. It is also
clear that there is at least one internal cleavage site in RAP
giving rise to the two lower bands appearing in these lanes, i.e.
the final product is cleaved into two pieces both visible on the
gel (3). Lanes F and G and lanes K and L show essentially the same
as lanes C and D, though the samples were taken later, after 23 and
26 hours of incubation with GrB--H6 giving rise to more cleavage in
the apparent internal site in RAP.
[0139] In lanes H and I is shown purified samples of H6-IEGR--RAP
cleaved partly (lane H) or completely (lane I) by FX.sub.a to give
the final RAP product. In these lanes the degradation products from
any internal cleavage by FX.sub.a has been removed by
purification.
[0140] FIG. 11 shows the SDS PAGE of the samples from the
H6-IEPD-RAP+GrB--H6 and the H6-IEGR--RAP+FX.sub.a incubations.
Description of the lanes (A-O):
[0141] A: Molecular weight marker
[0142] B: H6-IEGR--RAP alone after 27 hours incubation
[0143] C: 400 .mu.l H6-IEGR--RAP+1 .mu.l FX.sub.a after 72 hour
incubation
[0144] D: 400 .mu.l H6-IEGR--RAP+1 .mu.l FX.sub.a after 1 hour
incubation
[0145] E: 400 .mu.l H6-IEGR--RAP+1 .mu.l FX.sub.a after 3 hours
incubation
[0146] F: 400 .mu.l H6-IEGR--RAP+1 .mu.l FX.sub.a after 5 hours
incubation
[0147] G: 400 .mu.l H6-IEGR--RAP+1 .mu.l FX.sub.a after 7 hours
incubation
[0148] H: 400 .mu.l H6-IEGR--RAP+1 .mu.l FX.sub.a after 27 hours
incubation
[0149] I: H6-IEPD-RAP alone after 27 hours incubation
[0150] J: 400 .mu.l H6-IEPD-RAP+2 .mu.l GrB--H6 after 1/2 hour
incubation
[0151] K: 400 .mu.l H6-IEPD-RAP+2 .mu.l GrB--H6 after 1 hour
incubation
[0152] L: 400 .mu.l H6-IEPD-RAP+2 .mu.l GrB--H6 after 3 hours
incubation
[0153] M: 400 .mu.l H6-IEPD-RAP+2 .mu.l GrB--H6 after 5 hours
incubation
[0154] N: 400 .mu.l H6-IEPD-RAP+2 .mu.l GrB--H6 after 7 hours
incubation
[0155] O: 400 .mu.l H6-IEPD-RAP+2 .mu.l GrB--H6 after 27 hours
incubation
[0156] In lanes B-H are the samples from the H6-IEGR--RAP
incubation (1), where lane B shows non-cleaved H6-IEGR--RAP. Lane
C-H shows that after only 1/2 hour almost all of the fusion protein
has been cleaved by FX.sub.a to give the correct product. In lanes
D-G some degradation products show up, and in lane H after 27 hours
of incubation all of the fusion protein has been degraded to give a
variety of smaller pieces, and there is no correctly cleaved
product left.
[0157] Lanes I-O shows the samples from the H6-IEPD-RAP incubation
(2). Lane I shows non-cleaved H6-IEPD-RAP, and as for H6-IEGR--RAP
nearly all the H6-IEPD-RAP has been cleaved correctly after only
1/2 hour incubation with GrB--H6, as is seen in lane J. In lanes
K-N degradation products show up, but not nearly as many as for the
H6-IEGR--RAP incubation. In lane O after 27 hours of incubation
there is still quite a lot of correctly cleaved product left,
[0158] FIG. 12 shows the SDS PAGE of the samples from the
H6Ubi-IEPD-ApoA1+GrB--H6 C228F and the H6Ubi-IEGR-ApoA1+FX.sub.a
incubations. Description of the lanes (A-M):
[0159] A: Molecular weight marker
[0160] B: H6Ubi-IEPD-ApoA1 alone, 0 hours incubation
[0161] C: 400 .mu.g H6Ubi-IEPD-ApoA1+0.4 .mu.g GrB--H6 C228F, 1
hour incubation
[0162] D: 400 .mu.g H6Ubi-IEPD-ApoA1+0.4 .mu.g GrB--H6 C228F, 3
hour incubation
[0163] E: 400 .mu.g H6Ubi-IEPD-ApoA1+0.4 .mu.g GrB--H6 C228F, 6
hours incubation
[0164] F: 400 .mu.g H6Ubi-IEPD-ApoA1+0.4 .mu.g GrB--H6 C228F, 24
hours incubation
[0165] G: 400 .mu.g H6Ubi-IEPD-ApoA1+0.4 .mu.g GrB--H6 C228F, 48
hours incubation
[0166] H: H6Ubi-IEGR-ApoA1 alone, 0 hours incubation
[0167] I: 350 .mu.g H6Ubi-IEGR-ApoA1+0.35 .mu.g FX.sub.a, 1 hour
incubation
[0168] J: 350 .mu.g H6Ubi-IEGR-ApoA1+0.35 .mu.g FX.sub.a, 3 hour
incubation
[0169] K: 350 .mu.g H6Ubi-IEGR-ApoA1+0.35 .mu.g FX.sub.a, 6 hours
incubation
[0170] L: 350 .mu.g H6Ubi-IEGR-ApoA1+0.35 .mu.g FX.sub.a, 24 hours
incubation
[0171] M: 350 .mu.g H6Ubi-IEGR-ApoA1+0.35 .mu.g FX.sub.a, 48 hours
incubation
[0172] In lanes B-G are the samples from the H6Ubi-IEPD-ApoA1
incubation (1), where lane B shows the non-cleaved H6Ubi-IEPD-ApoA1
preparation. Lanes H-M shows the samples from the H6Ubi-IEGR-ApoA1
incubation (2), where lane H shows the non-cleaved H6Ubi-IEGR-ApoA1
preparation. The position of the intact, non-cleaved fusion
proteins is indicated by (3). The bands marked by (4) is the
correctly cleaved ApoA1 product, whereas the bands marked with (5)
H6Ubi fusion partner.
[0173] FIG. 13 shows the SDS PAGE of the samples from the
H6-IEPD-TN123+GrB--H6 incubation after 12 hours and 5 days without
addition of Ca.sup.2+. Some samples have been reduced. Description
of the lanes (A-N):
[0174] A: Molecular weight marker
[0175] B: 200 .mu.l H6-IEPD-TN123+1 .mu.l GrB--H6 after 5 days
incubation, sample reduced
[0176] C: 200 .mu.l H6-IEPD-TN123+10 .mu.l GrB--H6 after 5 days
incubation, sample reduced
[0177] D+E: H6-IEPD-TN123 alone after 5 days incubation
[0178] F: 200 .mu.l H6-IEPD-TN123+1 .mu.l GrB--H6 after 5 days
incubation
[0179] G: 200 .mu.l H6-IEPD-TN123+10 .mu.l GrB--H6 after 5 days
incubation
[0180] H: GrB--H6 alone diluted as in C and G
[0181] I: H6-IEPD-TN123 alone after 12 hours incubation
[0182] J: 200 .mu.l H6-IEPD-TN123+1 .mu.l GrB--H6 after 12 hours
incubation
[0183] K: 200 .mu.l H6-IEPD-TN123+10 .mu.l GrB--H6 after 12 hours
incubation
[0184] L: H6-IEPD-TN123 alone after 12 hours incubation, sample
reduced
[0185] M: 200 .mu.l H6-IEPD-TN123+1 .mu.l GrB--H6 after 12 hours
incubation, sample reduced
[0186] N: 200 .mu.l H6-IEPD-TN123+10 .mu.l GrB--H6 after 12 hours
incubation, sample reduced
[0187] Lanes I-K are identical to lanes F-H in FIG. 2 and I-K in
FIG. 3, i.e. samples after 12 hours incubation with either 0, 0.2
or 2 .mu.g GrB--H6. Here the band pattern (1) indicates an internal
cleavage site in the TN123 part of H6-IEPD-TN123, and the pattern
is further explained in FIG. 12. Lanes D-G show the incubations
after 5 days, and here most of the fusion protein has been cleaved.
In lane G (10 .mu.l GrB--H6 added) almost all of the fusion protein
has been cleaved twice (2); at the IEPD.dwnarw. sequence as well as
at the internal site in TN123 with the sequence AQPD.dwnarw..
[0188] Lanes L-N and lanes B-D show the same samples after 12 hours
and after 5 days, respectively, but here the samples are reduced.
This band pattern (3) is also explained in FIG. 12 and again almost
all of the fusion protein has been cleaved twice after 5 days with
10 .mu.l GrB--H6, lane C (4).
[0189] FIG. 14 shows the SDS PAGE of the samples from the
H6-IEPD-TN123+GrB--H6 incubation after 12 hours and 2 days with the
addition of 5 mM Ca.sup.2+. Some samples have been reduced.
Description of the lanes (A-K):
[0190] A: Molecular weight marker
[0191] B: 200 .mu.l H6-IEPD-TN123+1 .mu.l GrB--H6 and 5 mM
CaCl.sub.2, sample reduced
[0192] C: 200 .mu.l H6-IEPD-TN123+10 .mu.l GrB--H6 and 5 mM
CaCl.sub.2, sample reduced
[0193] D: H6-IEPD-TN123 alone
[0194] E: 200 .mu.l H6-IEPD-TN123+1 .mu.l GrB--H6 and 5 mM
CaCl.sub.2
[0195] F: 200 .mu.l H6-IEPD-TN123+1 .mu.l GrB--H6 and no
CaCl.sub.2
[0196] G: 200 .mu.l H6-IEPD-TN123+10 .mu.l GrB--H6 and 5 mM
CaCl.sub.2
[0197] H: 200 .mu.l H6-IEPD-TN123+10 .mu.l GrB--H6 and no
CaCl.sub.2
[0198] I: GrB--H6 alone diluted as in G and H
[0199] J: 200 .mu.l H6-IEPD-TN123+1 .mu.l GrB--H6 and no CaCl.sub.2
after 2 days incubation
[0200] K: 200 .mu.l H6-IEPD-TN123+10 .mu.l GrB--H6 and no
CaCl.sub.2 after 2 days incubation
[0201] Lanes B-H and J-K show the incubations of H6-IEPD-TN123 with
GrB--H6 after 12 hours and 2 days, respectively.
[0202] Lane D shows non-cleaved H6-IEPD-TN123. Comparing lane E and
G (+5 mM Ca.sup.2+) with lane F and H (no Ca.sup.2+) only two bands
appear with 5 mM Ca.sup.2+ present (1), while four bands appear (2)
when no Ca.sup.2+ in present, as described for FIGS. 2, 3, 10 and
12. After 12 hours incubation with 10 .mu.l GrB--H6 approximately
40% of the fusion protein has been correctly cleaved when Ca.sup.2+
is present (lane G), while the cleavage of the two sites when no
Ca.sup.2+ is present happens a bit faster (lane H after 12 hours
and K after 2 days). In lane K almost all the fusion protein has
been cleaved twice (3). The samples in lanes B and C are reduced
and still only two bands appear (4); the non-cleaved H6-IEPD-TN123
and the correctly cleaved product, where the H6 is removed.
[0203] FIG. 15 shows a schematic representation of the band pattern
observed on the SDS PAGE gels in FIGS. 2, 3, 10 and 11.
[0204] (A): When no Ca.sup.2+ is present the H6-IEPD-TN123
construct is cleaved at two different sites indicated by
"GrB--H6.fwdarw.". The small N-terminal part cleaved off is too
small to be visualized on the gel. The resulting molecule consists
of two polypeptide chains held together by a disulfide bond.
[0205] (1) and (2): In a non-reducing gel the band pattern in (2)
is obtained when the cleavage is not complete. (1) is the
non-cleaved H6-IEPD-TN123, and in (2) the remaining non-cleaved
fusion protein is the second band from the top. The top band in (2)
is H6-IEPD-TN123 cleaved at the internal site, AQPD.dwnarw., giving
a molecule of the same size as non-cleaved H6-IEPD-TN123, but less
compact. The band at the bottom is the correctly cleaved fusion
protein, whereas the third band from the top is the fusion protein
cleaved twice; both at the correct IEPD; site and at the internal
AQPD.dwnarw. site. When cleaved at the internal site the molecule
is less compact and therefore migrates shorter in the gel than the
correctly cleaved fusion protein.
[0206] (3) and (4): If the samples are reduced the band pattern in
(4) is observed. Here any disulfide bonds are broken, so only
single polypeptide chains are seen in the gel. (3) shows the
position of the non-cleaved, reduced H6-IEPD-TN123. The top band in
(4) is the remaining non-cleaved H6-IEPD-TN123, while the second
band from the top is the correctly cleaved and reduced
H6-IEPD-TN123. Under the reducing conditions the molecules cleaved
at the internal site are no longer held together by any disulfide
bonds, and only the larger one of the two polypeptides after
internal cleavage can be seen in the gel. Therefore the third band
from the top is the larger part of the internally cleaved fusion
protein, and the bottom band is this larger part after cleavage at
both the internal site and at the correct IEPD.dwnarw. site.
[0207] (B): When 5 mM Ca.sup.2+ is added to the incubations, no
internal cleavage is observed. Ca.sup.2+ ions bind to the
H6-IEPD-TN123 molecule in a way preventing GrB--H6 from cleaving
the fusion protein at the internal AQPD.dwnarw. site. With the
AQPD.dwnarw. site rendered inaccessible cleavage only occurs at the
correct IEPD.dwnarw. site.
[0208] (5) and (6): When only cleavage at the correct IEPD.dwnarw.
site occurs the band pattern in (6) is seen. The position of the
non-cleaved H6-IEPD-TN123 is shown in (5), and so the top band in
(6) is the remaining non-cleaved H6-IEPD-TN123. The bottom band is
the fusion protein cleaved only once at the correct site. The small
N-terminal peptide is too small to be visualized in the gel.
[0209] FIG. 16 shows samples from the incubations of three of the
five H6-TripUB variants with GrB--H6. The three variants are
H6-TripUB IEPD.dwnarw.SP, H6-TripUB IQAD.dwnarw.SP and H6-TripUB
IQAD.dwnarw.SG. Description of the lanes (A-P):
[0210] A: Molecular weight marker
[0211] B: H6-TripUB IEPD.dwnarw.SP alone after 24 hours
incubation
[0212] C: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6 after
2 hours incubation
[0213] D: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6 after
6 hours incubation
[0214] E: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6 after
24 hours incubation
[0215] F: 200 .mu.l H6-TripUB IEPD.dwnarw.SP+5 .mu.l GrB--H6 after
48 hours incubation
[0216] G: H6-TripUB IQAD.dwnarw.SP alone after 24 hours
incubation
[0217] H: 200 .mu.l H6-TripUB IQAD.dwnarw.SP+5 .mu.l GrB--H6 after
2 hours of incubation
[0218] I: 200 .mu.l H6-TripUB IQAD.dwnarw.SP+5 .mu.l GrB--H6 after
6 hours of incubation
[0219] J: 200 .mu.l H6-TripUB IQAD.dwnarw.SP+5 .mu.l GrB--H6 after
24 hours of incubation
[0220] K: 200 .mu.l H6-TripUB IQAD.dwnarw.SP+5 .mu.l GrB--H6 after
48 hours of incubation
[0221] L: H6-TripUB IQAD.dwnarw.SG alone after 24 hours
incubation
[0222] M: 200 .mu.l H6-TripUB IQAD.dwnarw.SG+5 .mu.l GrB--H6 after
2 hours incubation
[0223] N: 200 .mu.l H6-TripUB IQAD.dwnarw.SG+5 .mu.l GrB--H6 after
6 hours incubation
[0224] O: 200 .mu.l H6-TripUB IQAD.dwnarw.SG+5 .mu.l GrB--H6 after
24 hours incubation
[0225] P: 200 .mu.l H6-TripUB IQAD>SG+5 .mu.l GrB--H6 after 48
hours incubation
[0226] In lane B is shown non-cleaved H6-TripUB IEPD.dwnarw.SP,
while more and more correctly cleaved product appear in lanes C-F
after incubation with GrB--H6. In lane F after 48 hours incubation
approximately 2/3 of the original amount of non-cleaved H6-TripUB
IEPD.dwnarw.SP has been correctly cleaved. In lanes G-K is shown
the H6-TripUB IQAD.dwnarw.SP samples giving more or less the same
picture as for H6-TripUB IEPD.dwnarw.SP with non-cleaved H6-TripUB
IQAD.dwnarw.SP in lane G and an increasing amount of correctly
cleaved product in lanes H-K. The cleavage, though, is much slower
than cleavage of the IEPD.dwnarw.SP sequence and only a small
amount has been cleaved after 48 hours incubation. For the
H6-TripUB IQAD.dwnarw.SG samples in lanes L-P it is evident that
the cleavage is very much faster than for both H6-TripUB
IEPD.dwnarw.SP and H6-TripUB IQAD.dwnarw.SP. Lane L shows the
non-cleaved H6-TripUB IQAD.dwnarw.SG and already after only 2 hours
incubation the majority of the fusion protein has been cleaved to
give the correct product.
[0227] FIG. 17 shows samples from the incubations of two of the
five H6-TripUB variants with GrB--H6. The two remaining variants
are H6-TripUB VGPD.dwnarw.SP and H6-TripUB VGPD.dwnarw.FG.
Description of the lanes (A-K):
[0228] A: H6-TripUB VGPD.dwnarw.SP alone after 24 hours
incubation
[0229] B: 200 .mu.l H6-TripUB VGPD.dwnarw.SP+5 .mu.l GrB--H6 after
2 hours incubation
[0230] C: 200 .mu.l H6-TripUB VGPD.dwnarw.SP+5 .mu.l GrB--H6 after
6 hours incubation
[0231] D: 200 .mu.l H6-TripUB VGPD.dwnarw.SP+5 .mu.l GrB--H6 after
24 hours incubation
[0232] E: 200 .mu.l H6-TripUB VGPD.dwnarw.SP+5 .mu.l GrB--H6 after
48 hours incubation
[0233] F: H6-TripUB VGPD.dwnarw.FG alone after 24 hours
incubation
[0234] G: 200 .mu.l H6-TripUB VGPD.dwnarw.FG+5 .mu.l GrB--H6 after
2 hours incubation
[0235] H: 200 .mu.l H6-TripUB VGPD.dwnarw.FG+5 .mu.l GrB--H6 after
6 hours incubation
[0236] I: 200 .mu.l H6-TripUB VGPD.dwnarw.FG+5 .mu.l GrB--H6 after
24 hours incubation
[0237] J: 200 .mu.l H6-TripUB VGPD.dwnarw.FG+5 .mu.l GrB--H6 after
48 hours incubation
[0238] K: Molecular weight marker
[0239] In lane A is non-cleaved H6-TripUB VGPD.dwnarw.SP and in
lanes B-E more and more correctly cleaved product appears as seen
for H6-TripUB IEPD.dwnarw.SP (lanes B-F FIG. 16). Approximately
half the amount of fusion protein has been cleaved after 48 hours.
In lane F non-cleaved H6-TripUB VGPD.dwnarw.FG is shown and in
lanes G-J the correctly cleaved product of H6-TripUB VGPD.dwnarw.FG
appears. After only 2 hours incubation all the fusion protein has
been correctly cleaved.
[0240] FIG. 18 shows samples from the incubations of H6-TripUB
IEPD.dwnarw.SP, H6-TripUB IEPD.dwnarw.TQ and H6-TripUB
IEPD.dwnarw.IV with GrB--H6 C228F in the protease:fusion protein
ratio of 1:500 at 23.degree. C.
[0241] Description of lanes A-M:
[0242] A: Molecular weight marker
[0243] B: H6-TripUB IEPD.dwnarw.SP, 0 hours incubation
[0244] C: 250 .mu.l H6-TripUB IEPD.dwnarw.SP+1 .mu.l GrB--H6 C228F,
4 hours incubation
[0245] D: 250 .mu.l H6-TripUB IEPD.dwnarw.SP+1 .mu.l GrB--H6 C228F,
24 hours incubation
[0246] E: 250 .mu.l H6-TripUB IEPD.dwnarw.SP+1 .mu.l GrB--H6 C228F,
96 hours incubation
[0247] F: H6-TripUB IEPD.dwnarw.TQ, 0 hours incubation
[0248] G: 250 .mu.l H6-TripUB IEPD.dwnarw.TQ+1 .mu.l GrB--H6 C228F,
4 hours incubation
[0249] H: 250 .mu.l H6-TripUB IEPD.dwnarw.TQ+1 .mu.l GrB--H6 C228F,
24 hours incubation
[0250] I: 250 .mu.l H6-TripUB IEPD.dwnarw.TQ+1 .mu.l GrB--H6 C228F,
96 hours incubation
[0251] J: H6-TripUB IEPD.dwnarw.IV, 0 hours incubation
[0252] K: 250 .mu.l H6-TripUB IEPD.dwnarw.IV+1 .mu.l GrB--H6 C228F,
4 hours incubation
[0253] L: 250 .mu.l H6-TripUB IEPD.dwnarw.IV+1 .mu.l GrB--H6 C228F,
24 hours incubation
[0254] M: 250 .mu.l H6-TripUB IEPD.dwnarw.IV+1 .mu.l GrB--H6 C228F,
96 hours incubation
[0255] In lanes B-E the cleavage of H6-TripUB IEPD.dwnarw.SP is
shown (1), where the cleavage is almost 100% complete after 96
hours. Lanes F-I are the cleavage of H6-TripUB IEPD.dwnarw.TQ (2),
and this is app. 100% completed after only 24 hours. This is also
the case for the cleavage of H6-TripUB IEPD.dwnarw.IV (3) shown in
lanes J-M. The bands for the intact and cleaved H6-TripUB
IEPD.dwnarw.TQ are all positioned slightly lower in the gel, than
the bands for H6-TripUB IEPD.dwnarw.SP, since H6-TripUB
IEPD.dwnarw.TQ is a deletion mutant of H6-TripUB IEPD.dwnarw.SP.
The bands for H6-TripUB IEPD.dwnarw.IV, another deletion mutant,
are positioned even lower, since the deletion is larger than the
one in H6-TripUB IEPD.dwnarw.TQ.
[0256] FIG. 19, Panel A shows samples from the incubations of
H6-TripUB IEPD.dwnarw.SP and H6-TripUB IEPD.dwnarw.EP with GrB--H6
C228F in the protease:fusion protein ratio of 1:500 in a total
volume of 200 .mu.l at both 21.degree. C. and 37.degree. C.
[0257] Description of lanes A-O:
[0258] A: Molecular weight marker
[0259] B: H6-TripUB IEPD.dwnarw.SP, 0 hours incubation
[0260] C: 24 .mu.g H6-TripUB IEPD.dwnarw.SP+0.048 .mu.g GrB--H6
C228F, 21.degree. C., 4 hours
[0261] D: 24 .mu.g H6-TripUB IEPD.dwnarw.SP+0.048 .mu.g GrB--H6
C228F, 21.degree. C., 24 hours
[0262] E: 24 .mu.g H6-TripUB IEPD.dwnarw.SP+0.048 .mu.g GrB--H6
C228F, 21.degree. C., 48 hours
[0263] F: 24 .mu.g H6-TripUB IEPD.dwnarw.SP+0.048 .mu.g GrB--H6
C228F, 37.degree. C., 4 hours
[0264] G: 24 .mu.g H6-TripUB IEPD.dwnarw.SP+0.048 .mu.g GrB--H6
C228F, 37.degree. C., 24 hours
[0265] H: 24 .mu.g H6-TripUB IEPD.dwnarw.SP+0.048 .mu.g GrB--H6
C228F, 37.degree. C., 48 hours
[0266] I: H6-TripUB IEPD.dwnarw.EP, 0 hours incubation
[0267] J: 36 .mu.g H6-TripUB IEPD.dwnarw.EP+0.072 .mu.g GrB--H6
C228F, 21.degree. C., 4 hours
[0268] K: 36 .mu.g H6-TripUB IEPD.dwnarw.EP+0.072 .mu.g GrB--H6
C228F, 21.degree. C., 24 hours
[0269] L: 36 .mu.g H6-TripUB IEPD.dwnarw.EP+0.072 .mu.g GrB--H6
C228F, 21.degree. C., 48 hours
[0270] M: 36 .mu.g H6-TripUB IEPD.dwnarw.EP+0.072 .mu.g GrB--H6
C228F, 37.degree. C., 4 hours
[0271] N: 36 .mu.g H6-TripUB IEPD.dwnarw.EP+0.072 .mu.g GrB--H6
C228F, 37.degree. C., 24 hours
[0272] O: 36 .mu.g H6-TripUB IEPD.dwnarw.EP+0.072 .mu.g GrB--H6
C228F, 37.degree. C., 48 hours
[0273] The intact, non-cleaved H6-TripUB IEPD.dwnarw.SP is shown in
lane B (1), while the cleavages at 21.degree. C. and 37.degree. C.
are shown in lanes C-E (2) and lanes F-H (3), respectively. There
is almost no difference in the two temperatures. After 48 hours
approximately 40% has been cleaved at 21.degree. C. Non-cleaved
H6-TripUB IEPD.dwnarw.EP is shown in lane I (4), while the cleavage
reactions at 21.degree. C. and 37.degree. C. are shown in lanes J-L
(5) and lanes M-O (6), respectively. Again almost no difference
between the two temperature, and after 48 hours at 21.degree. C.
approximately 35-40% has been cleaved. i.e. the GrB--H6 C228F
cleaves both substrates equally well.
[0274] FIG. 19, Panel B shows samples from the incubations of
H6-TripUB IEPD.dwnarw.EG and H6-TripUB IEPD.dwnarw.EP with GrB--H6
C228F in the protease:fusion protein ratio of 1:500 in a total
volume of 200 .mu.l at both 22.degree. C. and 37.degree. C.
[0275] Description of lanes A-O:
[0276] A: Molecular weight marker
[0277] B: H6-TripUB IEPD.dwnarw.EG, 0 hours incubation
[0278] C: 40 .mu.g H6-TripUB IEPD.dwnarw.EG+0.08 .mu.g GrB--H6
C228F, 23.degree. C., 6 hours
[0279] D: 40 .mu.g H6-TripUB IEPD.dwnarw.EG+0.08 .mu.g GrB--H6
C228F, 23.degree. C., 24 hours
[0280] E: 40 .mu.g H6-TripUB IEPD.dwnarw.EG+0.08 .mu.g GrB--H6
C228F, 23.degree. C., 50 hours
[0281] F: 40 .mu.g H6-TripUB IEPD.dwnarw.EG+0.08 .mu.g GrB--H6
C228F, 37.degree. C., 6 hours
[0282] G: 40 .mu.g H6-TripUB IEPD.dwnarw.EG+0.08 .mu.g GrB--H6
C228F, 37.degree. C., 24 hours
[0283] H: 40 .mu.g H6-TripUB IEPD.dwnarw.EG+0.08 .mu.g GrB--H6
C228F, 37.degree. C., 50 hours
[0284] I: H6-TripUB IEPD.dwnarw.EP, 0 hours incubation
[0285] J: 38 .mu.g H6-TripUB IEPD.dwnarw.EP+0.08 .mu.g GrB--H6
C228F, 23.degree. C., 6 hours
[0286] K: 38 .mu.g H6-TripUB IEPD.dwnarw.EP+0.08 .mu.g GrB--H6
C228F, 23.degree. C., 24 hours
[0287] L: 38 .mu.g H6-TripUB IEPD.dwnarw.EP+0.08 .mu.g GrB--H6
C228F, 23.degree. C., 50 hours
[0288] M: 38 .mu.g H6-TripUB IEPD.dwnarw.EP+0.08 .mu.g GrB--H6
C228F, 37.degree. C., 6 hours
[0289] N: 38 .mu.g H6-TripUB IEPD.dwnarw.EP+0.08 .mu.g GrB--H6
C228F, 37.degree. C., 24 hours
[0290] O: 38 .mu.g H6-TripUB IEPD.dwnarw.EP+0.08 .mu.g GrB--H6
C228F, 37.degree. C., 50 hours
[0291] The intact, non-cleaved H6-TripUB IEPD.dwnarw.EG is shown in
lane B (1), while the cleavages at 21.degree. C. and 37.degree. C.
are shown in lanes C-E (2) and lanes F-H (3), respectively. There
is almost no difference in the two temperatures, and after only 6
hours the cleavage has been completed. Non-cleaved H6-TripUB
IEPD.dwnarw.EP is shown in lane I (4), while the cleavage reactions
at 21.degree. C. and 37.degree. C. are shown in lanes J-L (5) and
lanes M-O (6), respectively. As in FIG. 19 Panel A after 50 hours
at 21.degree. C. approximately 40% has been cleaved. Surprisingly
the GrB--H6 C228F cleaves the IEPD.dwnarw.EG substrate much better
than IEPD.dwnarw.SP and IEPD.dwnarw.EP.
[0292] FIG. 20 shows samples from the incubation of either
H6-TripUB IQAD.dwnarw.SP or H6-TripUB IQAD.dwnarw.SG with the six
different preparations of immobilized GrB--H6 C228F, experiment
A-F, described in Example 9.
[0293] Description of lanes A-M:
[0294] A: H6-TripUB IQAD.dwnarw.SP+immobilized GrB--H6 C228F,
experiment A
[0295] B: H6-TripUB IQAD.dwnarw.SP+immobilized GrB--H6 C228F,
experiment B
[0296] C: H6-TripUB IQAD.dwnarw.SP+immobilized GrB--H6 C228F,
experiment C
[0297] D: H6-TripUB IQAD.dwnarw.SP+immobilized GrB--H6 C228F,
experiment D
[0298] E: H6-TripUB IQAD+SP+immobilized GrB--H6 C228F, experiment
E
[0299] F: H6-TripUB IQAD.dwnarw.SP+immobilized GrB--H6 C228F,
experiment F
[0300] G: H6-TripUB IQAD.dwnarw.SG+immobilized GrB--H6 C228F,
experiment A
[0301] H: H6-TripUB IQAD.dwnarw.SG+immobilized GrB--H6 C228F,
experiment B
[0302] I: H6-TripUB IQAD.dwnarw.SG+immobilized GrB--H6 C228F,
experiment C
[0303] J: H6-TripUB IQAD.dwnarw.SG+immobilized GrB--H6 C228F,
experiment D
[0304] K: H6-TripUB IQAD.dwnarw.SG+immobilized GrB--H6 C228F,
experiment E
[0305] L: H6-TripUB IQAD.dwnarw.SG+immobilized GrB--H6 C228F,
experiment F
[0306] M: Molecular weight marker
[0307] The incubation with H6-TripUB IQAD.dwnarw.SP are shown in
lanes A-F (1), while the H6-TripUB IQAD.dwnarw.SG incubations are
shown in lanes G-L (2). The band representing non-cleaved fusion
protein (H6-TripUB IQAD.dwnarw.SP or H6-TripUB IQAD.dwnarw.SG) is
marked by (3) and the position of the bands for the correctly
cleaved products are marked by (4).
EXAMPLES
Example 1
[0308] Design and Construction of Human Granzyme B Expression
Vectors
[0309] In order to prepare inactive pro-Granzyme B constructs, a
sequence encoding activated human Granzyme B (E.C. 3.4.21.79), i.e.
from Ile21 (Ile16 in chymotrypsinogen numbering) to Tyr246, was
cloned into a pT7 cloning vector containing a hexa-His tag (H6)
C-terminally (pT7 C-term H6), resulting in the expression vector
pT7-IEGR-GrB--H6. The sequence, MGSIEGR, containing the blood
clotting factor X.sub.a (FX.sub.a) recognition sequence IEGR was
thereby placed just N-- terminally to Ile21 in Granzyme B providing
a FX.sub.a cleavage site between Arg (R) and Ile21. The resulting
fusion protein pro-Granzyme B containing the FX.sub.a recognition
sequence and the C-terminal hexa-His tag is in the following
referred to as pro-IEGR-GrB--H6 and is shown in SEQ ID NO:1.
[0310] In order to form self-activating pro-Granzyme B proteins,
the expression vectors pT7-IEPD-GrB--H6 and pT7-IEAD-GrB--H6 were
constructed, wherein the FX.sub.a recognition sequence of IEGR was
substituted with the Granzyme B recognition sites IEPD or IEAD,
respectively. The resulting self-activating GrB proteins are in the
following referred to as pro-IEPD-GrB--H6 and pro-IEAD-GrB--H6,
respectively and are shown in SEQ ID NO:2 and SEQ ID NO:3. The
design and cloning of the vectors is outlined in the following
section.
[0311] The, presence of a free cysteine at amino acid position 228
(using the established standard chymotrypsinogen amino acid
numbering system) in human Granzyme B, and in particular in the
above described Granzyme B proteins, pro-IEGR-GrB--H6,
pro-IEPD-GrB--H6, and pro-IEAD-GrB--H6, has the potential to cause
complications during the refolding process described in Example 2,
decrease stability of the activated enzyme and provide higher
non-enzymatic reactivity towards disulfide containing substrates.
Therefore a number of recombinant mutant proteins based on
pro-IEPD-GrB--H6 were generated in which the Cys228 amino acid
residue (chymotrypsinogen amino acid numbering) was substituted
with serine (S), alanine (A), threonine (T), valine (V), or
phenylalanine (F) by site-directed mutation of the construct
pT7-IEPD-GrB--H6, giving the expression vectors pT7-IEPD-GrB--H6
C228S, pT7-IEPD-GrB--H6 C228A, pT7-IEPD-GrB--H6 C228T,
pT7-IEPD-GrB--H6 C228V, and pT7-IEPD-GrB--H6 C228F, respectively.
The resulting mutant proteins are in the following referred to as
pro-IEPD-GrB--H6 C228S (SEQ ID NO 4), pro-IEPD-GrB--H6 C228A (SEQ
ID NO 5), pro-IEPD-GrB--H6 C228T (SEQ ID NO 6), pro-IEPD-GrB--H6
C228V (SEQ ID NO 7), and pro-IEPD-GrB--H6 C228F (SEQ ID NO 8),
respectively, and collectively referred to as the pro-IEPD-GrB--H6
C228X mutants. All of these mutants were constructed as
self-activating Granzyme B proteases.
[0312] Construction of the pT7 C-term H6 Cloning Vector
[0313] The cloning vector pT7 C-term H6, was constructed by
ligation of the DNA fragment made from the oligonucleotide primers
H6 C-term fw (SEQ ID NO: 9) and H6 C-term rev (SEQ ID NO: 10) into
an NcoI and EcoRI cut vector, pT7 (Christensen J H et al., 1991),
using standard procedures.
[0314] Cloning of Human Granzyme B into pT7 C-term H6 Cloning
Vector
[0315] The expression vector pT7-IEGR-GrB--H6, was constructed by
ligation of the BamHI and EcoRI restricted DNA fragment GrB EcoRI
amplified from a mixture of cDNA, isolated from human bone marrow,
human leukocyte, human lymphnodes, and lymphoma (Raji) cells
(Clontech Laboratories, Inc cat #7181-1, 7182-1, 7164-1, 7167-1)
(with the oligonucleotide primers GrBfw (SEQ ID NO: 11) and GrBrev
EcoRI (SEQ ID NO: 12)) into a BamHI and EcoRI cut vector, pT7
C-term H6, using standard procedures. Outlines of the resulting
nucleotide sequence of GrB EcoRI, is given as SEQ ID NO: 13.
[0316] Construction of Expression Vectors for Self-Activating Human
Granzyme B, pro-IEPD-GrB--H6 and pro-IEAD-GrB--H6
[0317] The expression vectors pT7-IEPD-GrB--H6 and pT7-IEAD-GrB--H6
encoding the self-activating pro-Granzyme B proteins, were
constructed by using the QuikChange.TM. Site-Directed Mutagenesis
Kit (STRATAGENE, Catalog #200518) according to the manufacturers'
protocol. The expression vector pT7-IEGR-GrB--H6 was used as
template. The oligonucleotide primers GrB GR--PD fw and GrB GR--PD
rev (SEQ ID NO: 14 and 15) were used for construction of
pT7-IEPD-GrB--H6 and the oligonucleotide primers GrB GR-AD fw and
GrB GR-AD rev (SEQ ID NO: 16 and 17) were used for construction of
pT7-IEAD-GrB--H6.
[0318] Construction of Expression Vectors for the Self-Activating
pro-IEPD-GrB--H6 C228X Mutants
[0319] The expression vectors pT7-IEPD-GrB--H6 C228X encoding the
self-activating pro-GrB--H6 C228X mutant proteins, were all
constructed by using the QuikChange.TM. Site-Directed Mutagenesis
Kit (STRATAGENE, Catalog #200518) according to the manufacturers'
protocol. The expression vector pT7-IEPD-GrB--H6 was used as
template. The degenerated oligonucleotide primers GrB SAT fw and
GrB SAT rev (SEQ ID NO: 18 and 19) were used for construction of
pT7-IEPD-GrB--H6 C228S, pT7-IEPD-GrB--H6 C228A, and
pT7-IEPD-GrB--H6 C228S, where D=A, G, or T and H=T. C, or A in the
GrB SAT fw and GrB SAT rev primer sequences shown in Table 1. The
degenerated oligonucleotide primers GrB VF fw and GrB VF rev (SEQ
ID NO: 20 and 21) were used for construction of pT7-IEPD-GrB--H6
C228V and pT7-IEPD-GrB--H6 C228F, where K=G or T and M=A or C in
the GrB VF fw and GrB VF rev primer sequences shown in Table 1.
TABLE-US-00001 TABLE 1 Oligonucleotide primers SEQ ID Primer
Nucleotide sequence NO. H6 C-term fw
5'-CATGGACGGAAGCTTGAATTCACATCACCATCACCATCACTA 9 ACGC-3' H6 C-term
rev 5'-AATTGCGTTAGTGATGGTGATGGTGATGTGAATTCAAGCTTC 10 CGCT-3' GrBfw
5'-CATGGGATCCATCGAGGGTAGGATCATCGGGGGACATG 11 AG-3' GrBrev EcoRI
5'-GCGTGAATTCAGGTACCGTTTCATGGTTTTCTTTATCC-3' 12 GrB GR-PD fw
5'-TCCATCGAGCCGGATATCATCGGGGGACATGAG-3' 14 GrB GR-PD rev
5'-CCCCGATGATATCCGGCTCGATGGATCCCATATG-3' 15 GrB GR-AD fw
5'-TCCATCGAGGCTGATATCATCGGGGGACATGAG-3' 16 GrB GR-AD rev
5'-CCCCGATGATATCAGCCTCGATGGATCCCATATG-3' 17 GrB SAT fw
5'-TCCACGAGCADCCACCMAGTCTCAAG-3' 18 GrB SAT rev
5'-AGACTTTGGTGGHGGCTCGTGGAGGC-3' 19 GrB VF fw
5'-TCCACGAGCCKTCACCAAAGTCTCAAG-3' 20 GrB VF rev
5'-AGACTTTGGTGAMGGCTCGTGGAGGC-3' 21
Example 2
[0320] Expression and Refolding of Self-Activating Human Granzyme
B
[0321] FX.sub.a Activateable Recombinant Pro-IEGR-GrB--H6
[0322] The FX.sub.a activateable recombinant pro-Granzyme B fusion
protein pro-IEGR-GrB--H6 (SEQ ID NO:1) was produced by growing and
expressing the expression vector pT7-IEGR-GrB--H6 prepared in
Example 1 in E. coli BL21 cells in a medium scale (3.times.1 litre)
as described by Studier F W et al. (1990). Exponentially growing
cultures at 37.degree. C. were at OD.sub.600=0.8 infected with
bacteriophage .lamda.CE6 at a multiplicity of approximately 5.
Cultures were grown at 37.degree. C. and 50 min after infection 0.1
g/L rifampicin (dissolved as 0.1 g/mL in methanol) was added. After
another three hours at 37.degree. C. the cells were harvested by
centrifugation. The cells were lysed by osmotic shock and
sonification and total cellular protein was extracted into phenol
(adjusted to pH 8 with Trisma base). The protein was precipitated
from the phenol phase by addition of 2.5 volumes of ethanol and
centrifugation. The protein pellet was dissolved in a buffer
containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8, and 100
mM dithiothreitol. Following gel-filtration on Sephadex.TM. G-25
Fine (Amersham Biosciences) into 8 M Urea, 0.5 M NaCl, 50 mM
Tris-HCl pH 8, and 5 mM 2-mercaptoethanol, the crude protein
preparation was applied onto a Ni.sup.2+-activated NTA-agarose
column (Ni.sup.2+-NTA-agarose, Quiagen).
[0323] Upon application of the crude protein extract onto the
Ni.sup.2+-NTA-agarose column, the fusion protein, pro-IEGR-GrB--H6
was purified from the majority of E. coli and A phage proteins by
washing with one column volume of the loading buffer followed by
one column volume of 8 M Urea, 0.5 M NaCl, 50 mM sodium phosphate
pH 6.3 and 5 mM 2-mercaptoethanol, 1/2 column volume of 6 M
guanidinium chloride, 50 mM Tris-HCl pH 8, and 5 mM
2-mercaptoethanol and finally 1/2 column volume of 8 M Urea, 0.5 M
NaCl, 50 mM Tris-HCl pH 8, and 3 mM reduced glutathione.
[0324] The pro-IEGR-GrB--H6 fusion protein was refolded on the
Ni.sup.2+-NTA-agarose column using the cyclic refolding procedure
described by Thogersen et al. (International Patent Application WO
9418227). The gradient manager profile is described in the below
Table 2 with 0.5 M NaCl, 50 mM Tris-HCl pH 8, 2 mM reduced
glutathione, and 0.2 mM oxidized glutathione as buffer A and 6 M
urea, 0.5 NaCl, 50 mM Tris-HCl pH 8, and 3 mM reduced glutathione
as buffer B. TABLE-US-00002 TABLE 2 Step Time (min) Flow (mL/min) %
A % B 1 0 2 100 0 2 45 2 100 0 3 46 2 0 100 4 52 2 0 100 5 60 2 100
0 6 105 2 100 0 7 106 2 4 96 8 113 2 4 96 9 120 2 100 0 10 165 2
100 0 11 166 2 8 92 12 172 2 8 92 13 180 2 100 0 14 225 2 100 0 15
226 2 10 90 16 232 2 10 90 17 240 2 100 0 18 285 2 100 0 19 286 2
12 88 20 292 2 12 88 21 300 2 100 0 22 345 2 100 0 23 346 2 14 86
24 352 2 14 86 25 360 2 100 0 26 405 2 100 0 27 406 2 16 84 28 412
2 16 84 29 420 2 100 0 30 465 2 100 0 31 466 2 18 82 32 472 2 18 82
33 480 2 100 0 34 525 2 100 0 35 526 2 20 80 36 532 2 20 80 37 540
2 100 0 38 585 2 100 0 39 586 2 22 78 40 592 2 22 78 41 600 2 100 0
42 645 2 100 0 43 646 2 24 76 44 652 2 24 76 45 660 2 100 0 46 705
2 100 0 47 706 2 30 70 48 713 2 30 70 49 720 2 100 0 50 765 2 100 0
51 766 2 35 65 52 772 2 35 65 53 780 2 100 0 54 825 2 100 0 55 826
2 40 60 56 832 2 40 60 57 840 2 100 0 58 885 2 100 0 59 886 2 45 55
60 892 2 45 55 61 900 2 100 0 62 945 2 100 0 63 946 2 50 50 64 952
2 50 50 65 960 2 100 0 66 1005 2 100 0 67 1006 2 55 45 68 1012 2 55
45 69 1020 2 100 0 70 1065 2 100 0 71 1066 2 60 40 72 1072 2 60 40
73 1080 2 100 0 74 1125 2 100 0 75 1126 2 60 40 76 1132 2 60 40 77
1140 2 100 0 78 1185 2 100 0 79 1186 2 60 40 80 1192 2 60 40 81
1200 2 100 0 82 1245 2 100 0 83 1246 2 65 35 84 1252 2 65 35 85
1260 2 100 0 86 1305 2 100 0 87 1306 2 65 35 88 1312 2 65 35 89
1319 2 100 0 90 1364 2 100 0 91 1365 2 65 35 92 1371 2 65 35 93
1378 2 100 0 94 1423 2 100 0
[0325] After completion of the cyclic refolding procedure, the
pro-IEGR-GrB--H6 fusion protein was eluted from the
Ni.sup.2+-NTA-agarose column with a buffer containing 0.5 M NaCl,
50 mM Tris-HCl pH 8, and 10 mM EDTA pH 8.
[0326] After elution from the Ni.sup.2+-NTA column the
pro-IEGR-GrB--H6 protein was diluted with 1 volumes of 50 mM
Tris-HCl pH 8.0 before the pH was adjusted to 7 with HCl. The
protein was then applied onto a SP Sepharose.TM. Fast Flow
(Amersham Biosciences) ion exchange column. The protein was eluted
over 10 column volumes with a linear gradient from 250 mM NaCl, 50
mM Tris-HCl pH 7.0 to 1 M NaCl, 50 mM Tris-HCl pH 7.0. Samples from
the elution profile appear as a single distinct band in SDS-PAGE
analysis and migrate with the anticipated molecular weight of 27.4
kDa for monomeric pro-IEGR-GrB--H6.
[0327] Self-Activating pro-IEPD-GrB--H6 and pro-IEAD-GrB--H6
[0328] The self-activating recombinant Granzyme B fusion proteins
pro-IEPD-GrB--H6 (SEQ ID NO:2) and pro-IEAD-GrB--H6 (SEQ ID NO:3)
were produced by expression from the vectors pT7-IEPD-GrB--H6 and
pT7-IEAD-GrB--H6 prepared in Example 1, where the expression,
refolding, and purification was performed essentially as described
for pro-IEGR-GrB--H6 above.
[0329] The self activation of the two enzymes, pro-IEPD-GrB--H6 and
pro-IEAD-GrB--H6 was followed as described in Example 3 below.
[0330] Self-Activating pro-IEPD-GrB--H6 C228X Mutants
[0331] All the pro-IEPD-GrB--H6 C228X mutants (SEQ ID NOS. 4, 5, 6,
7 and 8) were expressed from the pT7-IEPD-GrB--H6 C228X expression
vectors essentially as described for the expression of
pro-IEGR-GrB--H6 above. Refolding of the pro-IEPD-GrB--H6 C228X
mutants were also done essentially as described for
pro-IEGR-GrB--H6 above, and activation on a cation exchange column
was done as described above for pro-IEPD-GrB--H6 and
pro-IEAD-GrB--H6.
[0332] Purification and complete activation of the self-activating
pro-IEPD-GrB--H6 C228X mutants was done in only four hours by
applying the refolded protein, still in the pro-form, to a cation
exchange column, SP Sepharose.TM. Fast Flow (Amersham Biosciences),
washing for four hours with 250 mM NaCl, 50 mM TrisHCl, pH 7.0, and
finally eluting the activated protein with 750 mM NaCl, 50 mM
TrisHCl, pH 7.0. After elution the activated mutants are referred
to as GrB--H6 C228S, GrB--H6 C228A, GrB--H6 C228T, GrB--H6 C228V,
and GrB--H6 C228F.
[0333] Expression levels of the pro-IEPD-GrB--H6 C228X mutants were
similar to the pro-IEPD-GrB--H6 expression level. However,
refolding efficiency differed by op to 90% relative to that of
pro-IEPD-GrB--H6. One mutant, pro-IEPD-GrB--H6 C228S, had a very
low refolding efficiency and was therefore not analyzed further.
This is contrary to what would be expected as the obvious
conservative choice for substitution of a Cysteine residue would be
Serine, since Serine most closely resembles Cysteine of all the
amino acids naturally occurring in proteins, both in size,
hydrophilicity and chemically.
[0334] The refolding efficiency of three of the other mutants,
pro-IEPD-GrB--H6 C228A, pro-IEPD-GrB--H6 C228T, and
pro-IEPD-GrB--H6 C228V was similar to that of pro-IEPD-GrB--H6.
[0335] A highly interesting finding was that higher protein
recovery after refolding and purification was obtained for the
pro-IEPD-GrB--H6 C228F mutant, wherein Cysteine had been replaced
with Phenylalanine, than was the case for non-mutated
pro-IEPD-GrB--H6 comprising the Cys228 amino acid residue. Thus,
when 70 mg, estimated by Bradford assay (Coomassie.RTM. Plus
Protein Assay Reagent Kit, Pierce Biotechnology) using bovine serum
albumin as protein standard, of either the pro-IEPD-GrB--H6 C228F
or the non-mutated pro-IEPD-GrB--H6 was applied to the above
described refolding and purification procedure, the final yield
(protein recovery) was found to be 1.5% and 0.5%, respectively.
This clearly shows that the mutated pro-IEPD-GrB--H6 C228F provides
for improved final protein recovery yields. The reasoning for the
lower recovery yield may be that when the non-mutated
pro-IEPD-GrB--H6 protein was applied for purification and
activation by cation exchange chromatography the protein apparently
tended to precipitate and thus reduced the final yield (recovery)
of active enzyme. No significant precipitation was observed for
pro-IEPD-GrB--H6 C228F. Therefore, substitution of Cysteine 228
with Phenylalanine appears to be favourable for Granzyme B, in
particular for self-activating Granzyme B. This is highly
surprising, as the amino acid Phenylalanine is chemically very
dissimilar to Cysteine and would not normally be the choice for a
Cysteine substitution.
[0336] In the following examples we therefore focused on the C228F
mutant, pro-IEPD-GrB--H6 C228F, along with pro-IEGR-GrB--H6 for
comparison.
Example 3
[0337] Activation of pro-IEGR-GrB--H6 Fusion Protein using Purified
Bovine Factor X.sub.a and Self-Activation of pro-IEPD-GrB--H6 and
pro-IEAD-GrB--H6
[0338] Activation of pro-IEGR-GrB--H6 by Factor X.sub.a
[0339] A sample of monomeric inactive pro-IEGR-GrB--H6 produced as
described in Example 2, was taken directly from the eluate from the
SP Sepharose ion exchange. One mg of pro-IEGR-GrB--H6 (in app. 10
ml) was activated by the addition of 50 .mu.g FX.sub.a (50 .mu.l of
1 mg/ml) and incubated at room temperature for several days. The
degree of cleavage/activation of pro-IEGR-GrB--H6 by FX.sub.a,
resulting in GrB--H6, was estimated by SDS PAGE.
[0340] In addition, the Granzyme B activity during an incubation of
pro-IEGR-GrB--H6 with FX.sub.a was followed for several days using
the following colorimetric assay: 500 .mu.l buffer (100 mM NaCl, 50
mM Tris-HCl pH 8.0), 4 .mu.l 100 mM Ac--IEPD-pNA, and 5 .mu.l
incubation mixture. The incubation mixture was prepared by mixing
100 .mu.l pro-IEGR-GrB--H6 (approximately 10 .mu.g) with 1 .mu.l
FX.sub.a (1 mg/ml) and kept at 4.degree. C. during the incubation.
The results are summarised in Table 3 and FIG. 1. TABLE-US-00003
TABLE 3 Time Time (hours) .DELTA.OD.sub.405/min 0 hours 0 0.0073 2
hours 2 0.0200 5 hours 5 0.0250 19 hours 19 0.0829 2 days 45 0.1325
5 days 120 0.1832
[0341] To remove the added FX.sub.a the incubation mixture was
loaded onto a SP Sepharose.TM. Fast Flow (Amersham Biosciences) ion
exchange column washed in 250 mM NaCl, 50 mM Tris-HCl pH 7.0. The
FX.sub.a did not bind to the column material, while the resulting
GrB--H6 was eluted from the column with 750 mM NaCl, 50 mM Tris-HCl
pH 7.0.
[0342] Colorimetric Activity Measurements
[0343] To determine whether the added FX.sub.a had been properly
removed from the incubation mixture, both the GrB--H6 and the
FX.sub.a activity was measured before and after removal of the
added FX.sub.a using a colorimetric assay with the substrates S2222
(N-Benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p-nitroaniline,
Chromogenix, Italy, cat. no. S2222) and Ac--IEPD-pNA
(N-acetyl-L-isoleucyl-L-glutamyl-L-prolyl-L-aspartyl-p-nitroaniline,
Calbiochem, La Jolla, USA, cat. no. 368067), where the absorbance
was measured at 405 nm for approximately 3 minutes, and the
.DELTA.OD.sub.405/min was calculated. FX.sub.a activity was
measured using the following mix: 500 .mu.l buffer, 25 .mu.l 3 mM
S2222, and 5 .mu.l FX.sub.a. GrB--H6 activity was measured using
the following mix: 500 .mu.l buffer, 2 .mu.l 100 mM Ac--IEPD-pNA,
and 5 .mu.l GrB--H6.
[0344] The buffer used was either 100 mM NaCl, 50 mM Tris-HCl pH
8.0 or 100 mM HEPES pH 7.4. An example using the 100 mM NaCl, 50 mM
Tris-HCl pH 8.0 buffer is shown below in Table 3, where the top
fraction from the SP Sepharose eluate after FX.sub.a removal was
used: TABLE-US-00004 TABLE 4 Before FX.sub.a removal After FX.sub.a
removal (.DELTA.OD.sub.405/min) (.DELTA.OD.sub.405/min) GrB
activity FX.sub.a activity GrB activity FX.sub.a activity 0.1401
0.0139 0.2213 0.0001
[0345] As can be seen from the above Table 4, the added FX.sub.a
was completely removed from the activation mixture by ion exchange
on SP Sepharose.TM. Fast Flow column. The same result was obtained
with the buffer comprising 100 mM HEPES pH 7.4.
[0346] Self-Activation of pro-IEPD-GrB--H6 and pro-IEAD-GrB--H6
[0347] Recombinant self-activating human Granzyme B derivatives
IEPD-GrB--H6 and IEAD-GrB--H6 were produced as described in Example
2 by using the expression vectors pT7-IEPD-GrB--H6 and
pT7-IEAD-GrB--H6 described in Example 1. The IEAD-GrB--H6 and
IEPD-GrB--H6 proteins were eluted from the SP Sepharose columns and
stored at 4.degree. C. for 2 days before the activity of the
respective top fractions were determined by using a colorimetric
assay. For this purpose the following was mixed: 500 .mu.l buffer
(100 mM HEPES pH 7.5), 2 .mu.l 100 mM Ac--IEPD-pNA, and 5 .mu.l
protein solution. The change in absorption was then determined at
405 nm during 3 min. The activity was further determined after
additional incubation for 1 and 2 days at 4.degree. C. The results
are summarised in Table 5. TABLE-US-00005 TABLE 5 Protein
.DELTA.OD.sub.405/min IEAD-GrB-H6 2 days 0.1372 IEPD-GrB-H6 2 days
0.1284 IEAD-GrB-H6 3 days 0.1607 IEPD-GrB-H6 3 days 0.1375
IEAD-GrB-H6 4 days 0.1983 IEPD-GrB-H6 4 days 0.1351
[0348] As can be seen from the Table 5, the self-activating
derivatives pro-IEPD-GrB--H6 and pro-IEAD-GrB--H6 were activated
without addition of any previously activated Granzyme B and the
self-activation was not completed until after a least three or four
days at 4.degree. C.
Example 4
[0349] Granzyme B Activity Determined on Small Chromogenic Peptide
Substrate
[0350] The activity of the activated and purified GrB--H6 was
measured in different buffers using the Ac--IEPD-pNA substrate: 500
.mu.l buffer, 2 .mu.l 100 mM Ac--IEPD-pNA, and 5 .mu.l GrB--H6. The
.DELTA.OD.sub.405/min was calculated from the first 0.75 min unless
otherwise noted. TABLE-US-00006 TABLE 6 Approximate amount Of
GrB-H6 Activity Buffer added (.mu.g) (.DELTA.OD.sub.405/min) TN pH
8.1 1 0.2213 (1 min) TN pH 7.0 1 0.2794 TN pH 7.4 1 0.2930 0.2624
0.5 0.0835 0.1082 0.2 0.0245 (3 min) TN pH 7.4 + 0.1% TWEEN20 0.5
0.0887 0.2 0.0303 (3 min) TN pH 7.4 + 5 mM Ca.sup.2+ 0.5 0.1401 TN
pH 7.4 + 5 mM Mg.sup.2+ 0.5 0.1491 100 mM HEPES pH 7.5 0.5 0.2350
100 mM HEPES pH 7.5 + 5 mM Ca.sup.2+ 0.5 0.2425 100 mM HEPES pH 7.2
0.5 0.1970 100 mM HEPES pH 7.4 0.5 0.2273 0.2328 100 mM HEPES pH
7.4 + 50 mM KCl 0.5 0.2167 100 mM HEPES pH 7.4 + 50 mM NaCl 0.5
0.1993 0.1938 100 mM NaCl, 50 mM Tris-HCl pH 7.4 0.5 0.1682 50 mM
NaCl, 25 mM Tris-HCl pH 7.4 0.5 0.1948 100 mM KCl, 50 mM Tris-HCl
pH 7.4 0.5 0.1662 TN = 100 mM NaCl, 50 mM Tris-HCl
[0351] As can be seen from the above Table 6, 100 mM HEPES pH
7.4-7.5 was the best buffer for GrB--H6 activity of the buffers
evaluated.
[0352] In a later pH scanning experiment performed with GrB--H6
C228F the optimum pH for activity towards the substrate
Ac--IEPD-pNA was found to be in the range of pH 7.5-7.8 in 100 mM
HEPES.
[0353] To estimate the steady state kinetic parameters K.sub.M and
k.sub.cat, the same colorimetric assay as described above was used
with a total volume of 500 .mu.l in the assay cuvette. The assay
buffer was 100 mM HEPES pH 7.75, and both GrB--H6 and GrB--H6 C228F
were used in a concentration of 20 nM in each measurement. To
construct a Lineweaver-Burk plot the following substrate
concentrations were used: 5, 40, 150, 300, and 600 .mu.M. The
obtained results are shown in Table 7 below: TABLE-US-00007 TABLE 7
GrB-H6 GrB-H6 C228F K.sub.M (.mu.M) 66.9 27.0 k.sub.cat (s.sup.-1)
5.03 4.85 k.sub.cat/K.sub.M (10.sup.4 s.sup.-1M.sup.-1) 7.5
18.0
[0354] The obtained values for K.sub.M, k.sub.cat and
k.sub.cat/K.sub.M shown in the above Table 7 for GrB--H6 and
GrB--H6 C228F are very similar to the values found for a
recombinant rat GrB (Harris J. L. et al., 1998.)
Example 5
[0355] Estimation of the Specificity of GrB--H6 and GrB--H6 C228F
and the Stability of GrB--H6 C228F
[0356] The Specificity of GrB--H6 and GrB--H6 C228F
[0357] The specificity of both the GrB--H6 and the GrB--H6 C228F
protease was examined using the chromogenic substrates Ac-LEED-pNA,
Ac--VEID-pNA, Ac--YVAD-pNA, and Ac-DEVD-pNA, in addition to the
Ac--IEPD-pNA substrate applied in Example 4. The activity assay was
again carried out in 500 .mu.l 100 mM HEPES pH 7.75 with a
substrate concentration of 400 .mu.M. For each measurement 1 .mu.g
of protease was added to the assay cuvette. All measurements were
done in triplicate, and the activities obtained were normalized by
setting the activity measured on Ac--IEPD-pNA to 100%. The results
are shown in FIG. 2. It can be seen that the GrB--H6 protease is at
least as specific as the GrB--H6 C228F protease.
[0358] The Stability of GrB--H6 C228F
[0359] In order to determine the stability of the GrB--H6 C228F
protease, samples of GrB--H6 C228F was incubated in 100 mM HEPES pH
7.4 for 15 days at 4.degree. C., 23.degree. C. and 37.degree. C.
The 100 mM HEPES buffer was chosen in order to examine any
auto-cleavage and degradation, but no significant "cannibalism" was
observed as assessed by SDS PAGE (see FIG. 3A). The hydrolytic
activity towards the chromogenic substrate Ac--IEPD-pNA was also
measured during this incubation period, see FIG. 3B.
[0360] The GrB--H6 C228F protease is remarkably stable at 4.degree.
C. and 23.degree. C. The activity only falls slightly with
approximately 10% at 23.degree. C. during the 15 days, and almost
no degradation fragments are visible in the gel. Even at 37.degree.
C. there is still about 20% activity left after 15 days, and only
very few degradation fragments show up in the gel.
[0361] It has also been found that the GrB--H6 C228F protease on a
short time scale of 10 minutes is stable up to 50.degree. C. (not
shown here). In this experiment a sample of the protease was
incubated for 10 minutes at a given temperature and then returned
to room temperature by 10 minutes incubation at 23.degree. C. The
activity towards Ac--IEPD-pNA was then measured at 23.degree. C. Up
to the incubation temperature of 50.degree. C. the protease can
revert back to almost 100% activity after incubation at to room
temperature (23.degree. C.), but after exposure to a temperature
above 50.degree. C. the protease can no longer revert to an active
form, and only very little activity can be detected.
Example 6
[0362] Design and Construction of Expression Vectors for Fusions
Proteins Containing a Recognition Sequence Cleavable by GrB--H6 and
GrB--H6 C228F
[0363] In order to prepare suitable fusion proteins as substrates
for GrB--H6 and GrB--H6 C228F, the FX.sub.a recognition sequence in
the FX.sub.a cleavable fusion proteins H6-FX-TripBUB, H6-IEGR--RAP,
H6Ubi-IEGR-ApoA1, and H6-FX-TN123 (encoded by pT7H6-FX-TripBUB,
pT7H6-FX--RAP, pT7H6Ubi-FX-ApoA.sub.1, and pT7H6-FX-TN123,
respectively) was changed from either IEGR or IQGR to IEPD, giving
the constructs H6-TripUB IEPD.dwnarw.SP (SEQ ID NO. 22),
H6-IEPD-RAP (SEQ ID NO. 23), H6Ubi-IEPD-ApoA1 (SEQ ID NO. 24), and
H6-IEPD-TN123 (SEQ ID NO. 25).
[0364] In the construct H6-TripUB IEPD.dwnarw.SP the Granzyme B
recognition sequence is IEPD.dwnarw.SP, where .dwnarw. indicates
the cleavage site. This recognition sequence is located between the
H6 and the TripUB moiety of the construct, where the two residues,
SP, C-terminal to the scissile bond are the N-terminal part of the
TripUB moiety.
[0365] The recognition sequence in the following H6-TripUB fusion
proteins (termed the H6-TripUB variants) is indicated as the last
part of their name, as XXXX.dwnarw.YY, wherein XXXX is the part of
the Granzyme B recognition sequence between the hexa-His moiety H6
and the TripUB moiety, and wherein the YY residues are a part of
the TripUB moiety.
[0366] The IEPD.dwnarw.SP cleavage site in the H6-TripUB
IEPD.dwnarw.SP construct was changed to eight other cleavage sites
to give the following variants: H6-TripUB IQAD.dwnarw.SP (SEQ ID
NO. 26), H6-TripUB IQAD.dwnarw.SG (SEQ ID NO. 27), H6-TripUB
VGPD.dwnarw.SP (SEQ ID NO. 28), H6-TripUB VGPD.dwnarw.FG (SEQ ID
NO. 29), H6-TripUB IEPD.dwnarw.TQ (SEQ ID NO. 30), H6-TripUB
IEPD.dwnarw.IV (SEQ ID NO. 31), H6-TripUB IEPD.dwnarw.EP (SEQ ID
NO. 32), and H6-TripUB IEPD.dwnarw.EG (SEQ ID NO. 33), where
.dwnarw. indicates the cleavage site. In six of these eight
constructs the P.sub.1' and P.sub.2'-sites (both part of the TripUB
moiety) of the fusion protein were changed, namely in H6-TripUB
IQAD.dwnarw.SG, H6-TripUB VGPD.dwnarw.FG, H6-TripUB IEPD.dwnarw.TQ,
H6-TripUB IEPD.dwnarw.IV, H6-TripUB IEPD.dwnarw.EP, and H6-TripUB
IEPD.dwnarw.EG.
[0367] Construction of Fusion Protein Expression Vectors
[0368] The expression vector pT7H6-TripUB IEPD.dwnarw.SP was
constructed by using the QuikChange.TM. Site-Directed Mutagenesis
Kit (STRATAGENE, Catalog #200518) according to the manufacturers'
protocol with the vector pT7H6-FX-TripBUB (International Patent
Application WO 9856906) as template and the oligonucleotide
primers: TripUB GrB fw (SEQ ID NO: 34) and TripUB GrB rev (SEQ ID
NO: 35).
[0369] The expression vector pT7H6-IEPD-RAP was constructed by
site-directed mutagenesis as described above with the vector
pT7H6-FX--RAP (Nykjaer et al., 1992) as template and the
oligonucleotide primers: RAP GrB fw (SEQ ID NO: 36) and RAP GrB rev
(SEQ ID NO: 37).
[0370] The expression vector pT7H6Ubi-IEPD-ApoA1 was constructed by
site-directed mutagenesis as described above with the vector
pT7H6Ubi-FX-ApoA1 (International Patent Application WO0238609) as
template and the oligonucleotide primers: Mut-GrB fw (SEQ ID NO:
38) and Mut-GrB rw (SEQ ID NO: 39).
[0371] The expression vector pT7H6-IEPD-TN123 was constructed by
site-directed mutagenesis as described above with the vector
pT7H6-FX-TN123 (Holtet et al., 1997) as template and the
oligonucleotide primers: TN GrB fw (SEQ ID NO: 40) and TN GrB rev
(SEQ ID NO: 41).
[0372] The expression vector pT7H6-TripUB IQAD.dwnarw.SP was
constructed by using site-directed mutagenesis as described above
with the vector pT7H6-FX-TripBUB (WO 9856906) as template and the
oligonucleotide primers: PC7TripUB GR-AD fw (SEQ ID NO: 42) and
PC7TripUB GR-AD rev (SEQ ID NO: 43).
[0373] The expression vector pT7H6-TripUB IQAD.dwnarw.SG was
constructed by using site-directed mutagenesis as described above
with the vector pT7H6-TripUB IQAD.dwnarw.SP as template and the
oligonucleotide primers: PC7TripUB P-G fw (SEQ ID NO: 44) and
PC7TripUB P-G rev (SEQ ID NO: 45).
[0374] The expression vector pT7H6-TripUB VGPD.dwnarw.SP was
constructed by using site-directed mutagenesis as described above
with the vector pT7H6-TripUB IEPD.dwnarw.SP as template and the
oligonucleotide primers: DNATrip IE-VG fw (SEQ ID NO: 46) and
DNATrip IE-VG rev (SEQ ID NO: 47).
[0375] The expression vector pT7H6-TripUB VGPD.dwnarw.FG was
constructed by using site-directed mutagenesis as described above
with the vector pT7H6-TripUB VGPD.dwnarw.SP as template and the
oligonucleotide primers: DNATrip SP--FG fw (SEQ ID NO: 48) and
DNATrip SP--FG rev (SEQ ID NO: 49).
[0376] The expression vector pT7H6-TripUB IEPD.dwnarw.TQ was
constructed by a PCR reaction with the vector pT7H6-TripUB
IEPD.dwnarw.SP as template and the oligonucleotide primers: Trip
IEPD-TQ (SEQ ID NO: 50) and UB3 (SEQ ID NO: 52). The resulting PCR
product was digested with BamHI and HindIII and ligated into a
BamHI-HindII cut pT7H6(GS)3 vector (Christensen J. H et al.
1991).
[0377] The expression vector pT7H6-TripUB IEPD.dwnarw.IV was
constructed by a PCR reaction with the vector pT7H6-TripUB
IEPD.dwnarw.SP as template and the oligonucleotide primers: Trip
IEPD-IV (SEQ ID NO: 51) and UB3 (SEQ ID NO: 52). The resulting PCR
product was digested with BamHI and HindIII and ligated into a
BamHI-HindII cut pT7H6(GS)3 vector (Christensen J. H et al.
1991).
[0378] The expression vector pT7H6-TripUB IEPD.dwnarw.EP was
constructed by using site-directed mutagenesis as described above
with the vector pT7H6-TripUB IEPD.dwnarw.SP as template and the
oligonucleotide primers: TripUB EP fw (SEQ ID NO: 53) and TripUB EP
rev (SEQ ID NO: 54).
[0379] The expression vector pT7H6-TripUB IEPD.dwnarw.EG was
constructed by using site-directed mutagenesis as described above
with the vector pT7H6-TripUB IEPD.dwnarw.EP as template and the
oligonucleotide primers: TripUB EG fw (SEQ ID NO: 55) and TripUB EG
rev (SEQ ID NO: 56). TABLE-US-00008 TABLE 8 Oligonucleotide primers
SEQ ID Primer Nucleotide sequence NO. TripUB GrB fw
5'-GTGGATCCATCGAGCCTGACTCTCCTGGTACCGAGCC-3' 34 TripUB GrB rev
5'-GGTACCAGGAGAGTCAGGCTCGATGGATCCACTACCAC-3' 35 RAP GrB fw
5'-CGGATCCATCGAGCCTGACTACTCGCGGGAGAAG-3' 36 RAP GrB rev
5'-CCCGCGAGTAGTCAGGCTCGATGGATCCGTGATG-3' 37 Mut-GrB fw
5'-CGTGGTGGATCCATCGAGCCGGACGGTGGAGATGAACCC 38 CCC-3' Mut-GrB rw
5'-GGGGGGTTCATCTCCACCGTCCGGCTCGATGGATCCACC 39 ACG-3' TN GrB fw
5'-GGATCCATCGAGCCTGACGGCGAGCCACCAACC-3' 40 TN GrB rev
5'-GGCTCGCCGTCAGGCTCGATGGATCCGTGATGG-3' 41 PC7TripUB GR-AD fw
5'-GGATCCATCCAGGCAGACTCTCCTGGTACCGAG-3' 42 PC7TripUB GR-AD rev
5'-GTACCAGGAGAGTCTGCCTGGATGGATCCACTAC-3' 43 PC7TripUB P-G fw
5'-GGATCCATCCAGGCAGACTCTGGTGGTACCGAGCCAC-3' 44 PC7TripUB P-G rev
5'-CTCGGTACCACCAGAGTCTGCCTGGATGGATCCACTAC-3' 45 DNATrip IE-VG fw
5'-GTAGTGGATCAGTCGGGCCTGACTCTCCTGGTAC-3' 46 DNATrip IE-VG rev
5'-GAGAGTCAGGCCCGACTGATCCACTACCACTACC-3' 47 DNATrip SP-FG fw
5'-GGCCTGACTTTGGTGGTACCGAGCCACCAAC-3' 48 DNATrip SP-FG rev
5'-GGCTCGGTACCACCAAAGTCAGGCCCGACTG-3' 49 Trip IEPD-TQ
5'-GGGAAAGGATCCATCGAGCCTGACACCCAGAAGCCCAA 50 GAAGATTGTAAATG-3' Trip
IEPD-IV 5'-GGGAAAGGATCCATCGAGCCTGACATTGTAAATGCCAAG 51
AAAGATGTTGTGAAC-3' UB3
5'-CGCAAGCTTGCATGCTTAGGATCCACCACGAAGTCTCAA-3' 52 TripUB EP fw
5'-CGAGCCTGACGAGCCTGGTACCGAGCCAC-3' 53 TripUB EP rev
5'-CGGTACCAGGCTCGTCAGGCTCGATGGATC-3' 54 TripUB EG fw
5'-CCTGACGAGGGTGGTACCGAGCCACCAAC-3' 55 TripUB EG rev
5'-GCTCGGTACCACCCTCGTCAGGCTCGATG-3' 56
Example 7
[0380] Expression, Purification and Refolding of Fusion Proteins
Containing a Recognition Sequence Cleavable by GrB--H6 and GrB--H6
C228F
[0381] Expression of the Fusion Proteins
[0382] To prepare the chimeric fusion proteins H6-TripUB
IEPD.dwnarw.SP, H6-IEPD-RAP, H6-IEGR--RAP, H6Ubi-IEPD-ApoA1,
H6Ubi-IEGR-ApoA.sub.1, H6-IEPD-TN123 and the H6-TripUB variants,
the expression vectors pT7H6-TripUB IEPD.dwnarw.SP, pT7H6-IEPD-RAP,
pT7H6-FX--RAP, pT7H6Ubi-IEPD-ApoA1, pT7H6Ubi-IEGR-ApoA1,
pT7H6-IEPD-TN123, pT7H6-TripUB IQAD.dwnarw.SP, pT7H6-TripUB
IQAD.dwnarw.SG, pT7H6-TripUB VGPD.dwnarw.SP, pT7H6-TripUB
VGPD.dwnarw.FG, pT7H6-TripUB IEPD.dwnarw.TQ, pT7H6-TripUB
IEPD.dwnarw.IV, pT7H6-TripUB IEPD.dwnarw.EP, and pT7H6-TripUB
IEPD.dwnarw.EG (the last eight termed H6-TripUB variants) were
grown in a medium scale (3 litre; 2.times. TY medium, 5 mM
MgSO.sub.4 and 0.1 mg/ml ampicillin) in E. coli BL21 cells, as
described by Studier F W et al. (1990). Exponentially growing
cultures at 37.degree. C. were at OD.sub.600=0.8 infected with
bacteriophage .lamda.CE6 at a multiplicity of approximately 5.
Cultures were grown at 37.degree. C. for another four hours and the
cells harvested by centrifugation. Cells were re-suspended in 100
ml of 750 mM NaCl, 100 mM Tris-HCl pH 8, and 1 mM EDTA pH 8. Phenol
(150 ml adjusted to pH 8 with Trisma base) was added to each, and
the mixtures were sonicated to extract total protein. After
clarification by centrifugation (25 minutes at 10.000 g) crude
protein fractions were precipitated from the phenol phases by
addition of 2.5 volumes of 96% ethanol and centrifugation. Protein
pellets were dissolved in 75 ml 6 M guanidinium chloride, 50 mM
Tris-HCl pH 8, and 100 mM dithiothreitol (DTT).
[0383] Purification of H6-TripUB IEPD.dwnarw.SP, H6-IEPD-RAP.
H6-IEGR--RAP, H6Ubi-IEPD-ApoA1, H6Ubi-IEPD-ApoA1 and H6-TripUB
Variants
[0384] Following gel-filtration on Sephadex.TM. G-25 Fine (Amersham
Biosciences) into 8 M Urea, 500 mM NaCl, 50 mM Tris-HCl pH 8, and
10 mM 2-mercaptoethanol, the crude protein preparations of the
H6-IEPD-TripUB and H6-IEPD-RAP fusion proteins were applied by
batch adsorption onto Ni.sup.2+ activated NTA-agarose
(Ni.sup.2+-NTA-agarose, Quiagen) columns (usually 50-75 ml column
volume) for purification (Hochuli E et al., 1988). The column was
washed with the following: [0385] 1. 2.times. column volume 8 M
urea, 500 mM NaCl, 50 mM Tris-HCl pH 8, and 10 mM 2-mercaptoethanol
[0386] 2. 1.times. column volume 8 M urea, 500 mM NaCl, 50 mM
sodium-phosphate pH 6.3, and 10 mM 2-mercaptoethanol [0387] 3.
1.times. column volume 6 M guanidinium chloride, and 50 mM Tris-HCl
pH 8, and 10 mM 2-mercaptoethanol [0388] 4. 2.times. column volume
500 mM NaCl, and 50 mM Tris-HCl pH 8
[0389] The purified fusion proteins were then eluted with 500 mM
NaCl, 50 mM Tris-HCl pH 8, and 10 mM EDTA.
[0390] Purification and Refolding of H6-IEPD-TN123 Fusion
Proteins
[0391] Following gel-filtration on Sephadex.TM. G-25 Fine (Amersham
Biosciences) into 8 M Urea, 500 mM NaCl, 50 mM Tris-HCl pH 8, and
10 mM 2-mercaptoethanol, the crude protein preparations of the
H6-IEPD-TN123 fusion proteins were applied by batch adsorption to
Ni.sup.2+ activated NTA-agarose (Ni.sup.2+-NTA-agarose, Quiagen)
columns (usually 50-75 ml column volume) for purification and in
vitro refolding. The column was washed with the following: [0392]
1. 2.times. column volume 8 M urea, 500 mM NaCl, 50 mM Tris-HCl pH
8, and 10 mM 2-mercaptoethanol [0393] 2. 1.times. column volume 8 M
urea, 500 mM NaCl, 50 mM sodium-phosphate pH 6.3, and 10 mM
2-mercaptoethanol [0394] 3. 1.times. column volume 6 M guanidinium
chloride, 50 mM Tris-HCl pH 8, and 10 mM 2-mercaptoethanol
[0395] Each fusion protein was then subjected to the iterative
refolding procedure as described for plasminogen kringle 4 by
Thogersen et al. (International Patent Application WO 9418227).
After completion of the refolding procedure each refolded fusion
protein was then eluted from the Ni.sup.2+-NTA-agarose in 500 mM
NaCl, 50 mM Tris-HCl pH 8, 10 mM EDTA.
[0396] Fractions of each refolded fusion protein was gel filtrated
into 50 mM NaCl, 25 mM sodium acetate pH 5.0, and 1 mM CaCl.sub.2,
and was further purified by ion exchange chromatography on SP
Sepharose.TM. Fast Flow (Amersham Biosciences, 1.6 (i.d.) by 20
centimetre column) using a salt gradient from 50 mM NaCl, 25 mM
sodium acetate pH 5.0 and 1 mM CaCl.sub.2 to 1 M NaCl, 25 mM sodium
acetate pH 5.0, 1 mM CaCl.sub.2.
[0397] The final purification of each correctly folded fusion
protein product was then accomplished by gel-filtration into 25 mM
NaCl, 10 mM Tris-HCl pH 8, and 1 mM CaCl.sub.2 followed by ion
exchange chromatography on Q Sepharose.TM. Fast Flow (Amersham
Biosciences, 1.6 (i.d.) by 20 centimetre column) using a salt
gradient from 25 mM NaCl, 10 mM Tris-HCl pH 8, and 1 mM CaCl.sub.2
to 500 mM NaCl, 10 mM Tris-HCl pH 8, and 1 mM CaCl.sub.2.
Example 8
[0398] Cleavage of Prepared Fusion Proteins by GrB--H6, GrB--H6
C228F and FXa
[0399] Cleavage of H6-TripUB IEPD.dwnarw.SP by GrB--H6
[0400] The fusion protein H6-TripUB IEPD.dwnarw.SP (prepared as
described in Example 7) eluted from the Ni.sup.2+-NTA-agarose
column was gel filtrated into 100 mM HEPES pH 7.5 and 200 [l
samples of the top-fraction was incubated at room temperature with
either 0, 1 or 10 .mu.l of activated GrB--H6 (approximately 0, 0.2
and 2 .mu.g GrB--H6).
[0401] Samples for SDS PAGE were taken after 12, 19, and 24 hours
of incubation, and gels are shown in FIGS. 4 and 5.
[0402] Only the correctly cleaved product appear in lanes C-D in
FIG. 4 and lanes C-G in FIG. 5, and the longer the incubation time,
the more cleavage product appears in the lanes, both for the
addition of 1 and 10 .mu.l GrB--H6. The simple band pattern
observed is explained in FIG. 6. From this it is clear that GrB--H6
cleaved H6-TripUB IEPD.dwnarw.SP specifically at a single site.
Cleavage at the correct site after the IEPD sequence is confirmed
in lane E in FIG. 4, where the construct H6-FX-TripUB, containing
the FX.sub.a recognition site IQGR in place of the GrB recognition
site IEPD, was cleaved by FX.sub.a giving a product of the same
size as the GrB--H6 cleaved H6-TripUB IEPD.dwnarw.SP.
[0403] The Effect of Temperature and Addition of Ni.sup.2+ and NTA
on the Cleavage of H6-TripUB IEDP.dwnarw.SP by GrB--H6
[0404] With the H6-TripUB IEPD.dwnarw.SP fusion protein the
following nine incubations (Table 9) were set up using 200 .mu.l
H6-TripUB IEPD.dwnarw.SP and 5 .mu.l GrB--H6 (approximately 1 .mu.g
GrB--H6) for each incubation. TABLE-US-00009 TABLE 9 1 No addition
23.degree. C. 2 4.2 mM Ni.sup.2+ 3 4.2 mM Ni.sup.2+ + 5 mM NTA 4 No
addition 37.degree. C. 5 4.2 mM Ni.sup.2+ 6 4.2 mM Ni.sup.2+ + 5 mM
NTA 7 No addition 42.degree. C. 8 4.2 mM Ni.sup.2+ 9 4.2 mM
Ni.sup.2+ + 5 mM NTA
[0405] Samples for SDS PAGE were taken after 2, 7 and 22 hours of
incubation, see FIGS. 7, 8 and 9.
[0406] It was contemplated that the Ni.sup.2+ ions would bind the
N-terminal hexa-His tail (H6) of the fusion protein and facilitate
access to the cleavage site recognized by GrB--H6. In addition the
Ni.sup.2+ ions would also bind the C-terminal hexa-His tail of the
GrB--H6 construct. The addition of NTA was made to shield the
Ni.sup.2+ ions in solution in a similar fashion as on the
Ni.sup.2+-NTA agarose beads, i.e. to simulate the conditions on the
Ni.sup.2+-NTA agarose column.
[0407] FIG. 7 shows the incubations at 23.degree. C., FIG. 8 at
37.degree. C. and FIG. 9 at 42.degree. C. When no Ni.sup.2+ or NTA
was added, the H6-TripUB IEPD.dwnarw.SP fusion protein was cleaved
similar to what is seen in FIGS. 4 and 5, though after 22 hours it
seems that incubation at 37.degree. C. is the most optimal of the
three temperatures tested.
[0408] With the addition of 4.2 mM Ni.sup.2+ some protein
precipitated at the higher temperatures of 37.degree. C. and
42.degree. C., but no precipitation was seen at 230C. Because of
this no further cleavage of the fusion protein is seen in the gel
after 2 hours incubation at these two temperatures, where it seems
that both some H6-TripUB IEPD.dwnarw.SP and GrB--H6 precipitated.
At 23.degree. C. more fusion protein was cleaved after 22 hours
than with no addition of Ni.sup.2+.
[0409] The observed precipitation problem was eliminated by the
addition of 5 mM NTA to the incubations. After 22 hours of
incubation at 23.degree. C. more fusion protein had been cleaved
than with no Ni.sup.2+ or NTA addition, so the addition of
Ni.sup.2+ and NTA seems to speed up the cleavage reaction. By
further increasing the temperature to 37.degree. C. and 42.degree.
C. an even greater increase in the rate of cleavage is seen. After
22 hours of incubation at 37.degree. C. almost all the fusion
protein was cleaved to the correct product. A little less was
cleaved at 42.degree. C. after 22 hours.
[0410] Comparing the rate of cleavage initially estimated in the
experiments shown in FIGS. 4 and 5, to the rate of cleavage
observed here, it is clear that the addition of Ni.sup.2+ and NTA
as well as the incubation at 37.degree. C. dramatically speeds up
the specific cleavage of H6-TripUB IEPD.dwnarw.SP by GrB--H6.
[0411] Cleavage of H6-IEPD-RAP by GrB--H6
[0412] The fusion protein H6-IEPD-RAP (prepared as described in
Example 7) eluted from the Ni.sup.2+-NTA-agarose column was gel
filtrated into 100 mM HEPES pH 7.4 and 200 .mu.l samples of the
top-fraction was incubated at room temperature with either 0, 1 or
10 .mu.l of activated GrB--H6 (approximately 0, 0.2 and 2 .mu.g
GrB--H6). Samples for SDS PAGE were taken after 5, 23 and 26 hours
of incubation, see FIG. 10.
[0413] After only 5 hours incubation with either 1 or 10 .mu.l
GrB--H6 as described above all the H6-IEPD-RAP was cleaved to give
the final product. It is also clear that there is at least one
internal cleavage site in RAP, but this internal site was cleaved
much slower than the IEPD sequence, though. That GrB--H6 cleaved
off the H6 correctly at the IEPD sequence can be seen by comparing
the size of the product with purified samples of H6-FX--RAP cleaved
partly (lane H) or completely (lane I) by FX.sub.a to give the
final RAP product. In these lanes the degradation products from any
internal cleavage by FX.sub.a had been removed by purification.
[0414] Comparison of H6-IEPD-RAP Cleavage by GrB--H6 and
H6-IEGR--RAP Cleavage by FX.sub.a
[0415] The cleavage of H6-IEPD-RAP by GrB--H6 was compared with the
cleavage of H6-IEGR--RAP by FX.sub.a. Both H6-IEPD-RAP and
H6-IEGR--RAP were in 100 mM HEPES pH 7.4 and the following
incubations were set up at room temperature, 23.degree. C., with
the protease:fusion protein ratio 1:1000: [0416] 1. 400 .mu.l (app.
500 .mu.g) H6-IEGR--RAP+0.5 .mu.l FX.sub.a (1 mg/ml) (app. 0.5
.mu.g) [0417] 2. 400 .mu.l (app. 400 .mu.g) H6-GrB--RAP+2 .mu.l
GrB--H6 (app. 0.4 .mu.g)
[0418] Samples were taken for SDS PAGE after 0, 1/2, 1, 3, 5, 7 and
27 hours of incubation, see FIG. 11.
[0419] It is clear that both fusion proteins were cleaved very
rapidly by their respective protease. After only 1/2 hour almost
all of the fusion protein had been cleaved to give the correct
product for both incubations.
[0420] However, for the H6-IEGR--RAP+FX.sub.a incubation all of the
fusion protein had been degraded to give a variety of smaller
fragments after 27 hours, and there is no correctly cleaved product
left.
[0421] In the H6-IEPD-RAP+GrB--H6 incubation degradation fragments
are also seen, but not as many as for the H6-IEGR--RAP+FX.sub.a
incubation. There seems to be only one GrB-sensitive site in RAP
out of 19 possible sites (19 Asp residues in the RAP product),
while there are several FX.sub.a sensitive sites (26 possible
sites, 26 Arg residues). This slows down the degradation of
H6-IEPD-RAP by GrB--H6, whereby quite a lot of correctly cleaved
product (app. 25%) is still present after 27 hours of
incubation.
[0422] In summary the correct cleavage of the RAP fusion protein by
GrB--H6 is just as fast as by FX.sub.a, but the degradation of the
RAP fusion protein by GrB--H6 is much slower than the degradation
by FX.sub.a. In this example the GrB--H6 protease is therefore
superior to FX.sub.a, and it shows that GrB--H6 is a very specific
protease.
[0423] Comparison of H6Ubi-IEPD-ApoA1 Cleavage by GrB--H6 C228F and
H6Ubi-IEGR-ApoA1 cleavage by FX.sub.a
[0424] For the cleavage reactions of H6Ubi-IEPD-ApoA1 +GrB--H6
C228F and H6Ubi-IEGR-ApoA1+FX.sub.a, the protease:substrate ratio
was again 1:1000, and they were carried out at 23.degree. C. in 100
mM HEPES pH 7.76: [0425] 1. 250 .mu.l (app. 400 .mu.g)
H6Ubi-IEPD-ApoA1+0.4 .mu.g GrB--H6 C228F [0426] 2. 250 .mu.l (app.
350 .mu.g) H6Ubi-IEGR-ApoA1+0.35 .mu.g FX.sub.a
[0427] Samples were taken for SDS PAGE after 0, 1, 3, 6, 24 and 48
hours of incubation at 23.degree. C., see FIG. 12.
[0428] The GrB substrate, H6Ubi-IEPD-ApoA1, is approximately 100%
cleaved after only 6 hours incubation at 23.degree. C., whereas
only a small fraction of the FX.sub.a substrate, H6Ubi-IEGR-ApoA1,
has been cleaved after 6 hours. It is also seen that FX.sub.a
requires more than 48 hours to complete the cleavage of the
FX.sub.a substrate.
[0429] In the above two examples both the GrB--H6 and the GrB--H6
C228F protease is, as mentioned, superior to FX.sub.a, since it
either cleaves much faster (in the case of H6Ubi-X-ApoA1) or more
specific (in the case of H6-X--RAP) than the purified, bovine
FX.sub.a (where X denotes the recognition sites IEPD or IEGR). With
these two examples it is demonstrated that GrB--H6 and GrB--H6
C228F can both cleave off a short N-terminal tag like the hexa-His
tail (the H6 in H6-IEPD-RAP) and cleave between two protein domains
which are very closely connected by a short linker sequence
comprising the GrB cleavage site adjacent to the polypeptide of
interest (here the ApoA1 in H6Ubi-IEPD-ApoA1, with the linker
sequence GGSIEPD, wherein IEPD is the GrB recognition site). It was
verified that GrB--H6 and GrB--H6 C228F produced the correctly
cleaved products by N-terminal sequencing in both the above
cases.
[0430] Cleavage of H6-IEPD-TN123 by GrB--H6
[0431] The fusion protein H6-IEPD-TN123 (prepared as described in
Example 7) eluted from the Q Sepharose was after final purification
gel filtrated into 100 mM HEPES pH 7.5, and 200 .mu.l samples of
the top-fraction was incubated at room temperature with either 0, 1
or 10 .mu.l of activated GrB--H6 (approximately 0, 0.2 and 2 .mu.g
GrB--H6) both with and without 5 mM CaCl.sub.2 present. Samples for
SDS PAGE from the incubations without CaCl.sub.2 were taken after
12, 19 and 24 hours as well as 5 days of incubation. See FIGS. 4, 5
and 13. Samples for SDS PAGE from the incubations both with and
without CaCl.sub.2 were taken after approximately 20 and 48 hours
of incubation, see FIG. 14.
[0432] Without Ca.sup.2+:
[0433] The samples showed a distinct band pattern when
H6-IEPD-TN123 was cleaved by GrB--H6 with no Ca.sup.2+ present, as
seen in FIGS. 4, 5 and 13. The H6-IEPD-TN123 was cleaved correctly
at the IEPD sequence, but also just as rapidly at an internal site
of the sequence AQPD. The band pattern is explained in FIG. 15.
That H6-IEPD-TN123 was cleaved at the correct IEPD.dwnarw. site can
be seen from lane J in FIG. 4, where murine H6-FX-TN123 had been
cleaved by FX.sub.a giving a product of the same size as the
product from GrB--H6 cleavage of H6-IEPD-TN123 with no internal
cleavage, i.e. the lowest band of the four bands in the
pattern.
[0434] When the samples were reduced as in lanes B-D and L-N in
FIG. 13 a different band pattern appears. This pattern is also
explained in FIG. 15 and supports the notion of the specific
internal cleavage site AQPD.
[0435] With Ca.sup.2+:
[0436] FIG. 11 shows incubations of H6-IEPD-TN123 with GrB--H6,
where 5 mM CaCl.sub.2 were added to some of the incubations. Here
only two bands appear when Ca.sup.2+ is present (lanes E and G),
while four bands appear when no Ca.sup.2+ is present, as described
for FIGS. 4, 5, 13 and 15. This shows that by adding Ca.sup.2+ to
the incubation the internal cleavage site AQPD in Tetranectin
(TN123) can be made inaccessible to GrB--H6. This is because the
AQPD sequence is located in a loop, where the Q and D residues
participates in the binding of Ca.sup.2+-ions in Tetranectin.
Thereby only the correct cleavage at the specific IEPD site in the
fusion protein occurs, and the internal cleavage site in TN123 is
"turned off" by the addition of Ca.sup.2+.
[0437] Cleavage of H6-TripUB Variants
[0438] Each of the fusion proteins eluted from the
Ni.sup.2+-NTA-agarose column were gel filtrated into 100 mM HEPES
pH 7.4 and fractions of approximately the same concentration of the
five different H6-TripUB variants were used. The five variants were
H6-TripUB IEPD.dwnarw.SP, H6-TripUB IQAD.dwnarw.SP, H6-TripUB
IQAD.dwnarw.SG, H6-TripUB VGPD.dwnarw.SP and H6-TripUB
VGPD.dwnarw.FG. Of each fusion protein 200 .mu.l was incubated at
room temperature, 23.degree. C., with 5 .mu.l of activated GrB--H6
(approximately 1 .mu.g GrB--H6). The protease:fusion protein ratio
was thereby 1:500. Samples for SDS PAGE were taken after 2, 6, 24
and 48 hours of incubation and gels are shown in FIGS. 16 and
17.
[0439] In FIG. 16 are shown the samples of H6-TripUB IEPD.mu.SP,
H6-TripUB IQAD.mu.SP and H6-TripUB IQAD.dwnarw.SG. After 48 hours
incubation approximately 2/3 of the original amount of non-cleaved
H6-TripUB IEPD.dwnarw.SP had been correctly cleaved. In comparison
the cleavage of the sequence IQAD.dwnarw.SP, though, was much
slower than cleavage of the IEPD.dwnarw.SP sequence in H6-TripUB
IEPD.dwnarw.SP. No product is visible after 2 hours incubation and
only a small amount had been cleaved after 48 hours incubation.
From the H6-TripUB IQAD.dwnarw.SG samples it is evident that the
cleavage was much faster than for both H6-TripUB IEPD.dwnarw.SP and
H6-TripUB IQAD.dwnarw.SP. Already after 2 hours incubation the
majority of the fusion protein had been cleaved to give the correct
product. The single mutation of Pro (P) to Gly (G) in the P2' site
in the recognition sequence was enough for this dramatic change in
the cleavage rate.
[0440] In FIG. 17 are shown the samples from the H6-TripUB
VGPD.dwnarw.SP and H6-TripUB VGPD.dwnarw.FG incubations. The
cleavage of the VGPD.dwnarw.SP sequence was almost as fast as for
H6-TripUB IEPD.dwnarw.SP in FIG. 16. A small amount of product had
formed after 2 hours and approximately half the amount of fusion
protein had been cleaved after 48 hours. A dramatic change in
reaction rate occurred when the P1' and P2' sites were changed from
SP to FG in H6-TripUB VGPD.dwnarw.SP. After only 2 hours incubation
all the fusion protein had been correctly cleaved.
[0441] FIG. 18 shows the samples of the cleavage of H6-TripUB
IEPD.dwnarw.TQ and H6-TripUB IEPD.dwnarw.IV compared to H6-TripUB
IEPD.dwnarw.SP. The two constructs H6-TripUB IEPD.dwnarw.TQ and
H6-TripUB IEPD.dwnarw.IV are deletion mutants of the Trip part of
H6-TripUB IEPD.dwnarw.SP, where the first 7 residues are deleted in
H6-TripUB IEPD.dwnarw.TQ and the first 13 residues deleted in
H6-TripUB IEPD.dwnarw.IV.
[0442] Here the protease:fusion protein ratio was also 1:500, and
the reactions were performed at 23.degree. C. Samples for SDS PAGE
was taken after 4, 24, and 96 hours of incubation.
[0443] From the gel shown in FIG. 18 it is clear that the cleavage
of both H6-TripUB IEPD.dwnarw.TQ and H6-TripUB IEPD.dwnarw.IV is
much faster than for H6-TripUB IEPD.dwnarw.SP. As is shown in the
following FIG. 19 this may be because of the Pro in the P2' site of
H6-TripUB IEPD.dwnarw.SP.
[0444] FIG. 19, Panel A shows the cleavage of H6-TripUB
IEPD.dwnarw.SP and H6-TripUB IEPD.dwnarw.EP, while Panel B shows
the cleavage of H6-TripUB IEPD.dwnarw.EP and H6-TripUB
IEPD.dwnarw.EG. Here the protease:fusion protein ratio is again
1:500 and the cleavage reaction mixes were incubated both at
23.degree. C. and at 37.degree. C. For the gel in Panel A samples
for SDS PAGE were taken after 0, 4, 24 and 48 hours, and for the
gel in Panel B after 0, 6, 24 and 50 hours.
[0445] From these gels it is clear that a Pro in the P2' site is
disadvantageous for the rate of cleavage by GrB--H6 C228F. A
surprise, though, is that the GrB--H6 C228F is able to cleave the
substrate containing the IEPD.dwnarw.EP site. It cleaves this site
with the same low efficiency as the IEPD.dwnarw.SP site, but an
acidic residues like Glu (E) in the P.sub.1' site is infamous for
abolishing cleavage by most serine proteases, for example purified,
bovine FX.sub.a.
[0446] If we now consider the P.sub.1'P.sub.2' sites of
IEPD.dwnarw.SP, IQAD.dwnarw.SP, IQAD.dwnarw.SG, IEPD.dwnarw.EP, and
IEPD.dwnarw.EG it shows that the change in the P.sub.2' site from
Proline to Glyine enhances the cleavage dramatically. This
preference for G in the P.sub.2' position was also found by Harris
J L et al., 1998 for wt rat GrB using peptide substrates displayed
on phage particles. Again GrB--H6 C228F shows a surprisingly
effective cleavage of at the IEPD.dwnarw.EG site in spite of the E
in P.sub.1', which is, as mentioned above, is well-known in the art
for obstructing cleavage by most serine proteases. At a protease to
substrate ratio of 1:500 it takes less than 5 hours for GrB--H6
C228F to complete the cleavage of H6-TripUbi IEPD.dwnarw.EG. In
this respect the GrB--H6 C228F is again superior to FX.sub.a.
[0447] In addition to these observations it is also important to
note that even after 48 hours incubation with GrB--H6 or GrB--H6
C228F no internal cleavage took place in any of the H6-TripUB
variants, showing that GrB--H6 and GrB--H6 C228F cleaves very
specifically at the engineered recognition sites, even though the
TripUB sequence contains 7 other Asp (D) residues.
Example 9
[0448] Immobilization of Granzyme B
[0449] In order to be able to easily remove the Granzyme B enzyme
from the cleavage mixture the GrB--H6 C228F variant was used for
immobilization onto a gel matrix in six experiments as described in
the following.
[0450] The immobilization was performed in 0.3 M NaHCO.sub.3/NaOH,
pH 8.6 using the divinyl sulfone activated matrix called Mini-Leak
(Kem-En-Tec). Two levels of activation were used; 2-5 millimoles
and 10-50 millimoles vinyl groups per litre sedimented beads,
respectively, and for each level of activation three experiments
with different protein concentration and with or without PEG 20000
present was performed. The six experiments are summarized in Table
10. For the immobilization GrB--H6 C228F in 0.3 M NaHCO.sub.3/NaOH;
pH 8.6 was used at a protein concentration of 4 mg/ml, as estimated
from a Bradford assay using bovine serum albumin as protein
standard. The enzymatic activity of the GrB--H6 C228F solution was
measured as described example 4 using the buffers 0.3 M
NaHCO.sub.3/NaOH; pH 8.6 and 30% PEG 20000; 0.3 M NaHCO.sub.3 as
assay buffers. The immobilization was performed by mixing drained
gel, protein solution, and buffers to provide the volumes and
concentrations listed in Table 10 followed by mixing at room
temperature for 48 hours. TABLE-US-00010 TABLE 10 Protein
Activation concentration % PEG Efficiency Experiment level (mM)
(mg/ml) 20000 (%) A 2-5 0.75 0 10 B 2-5 0.75 9 18 C 2-5 2 0 10 D
10-50 0.75 0 16 E 10-50 0.75 9 10 F 10-50 2 0 10
[0451] Excess active groups were blocked by addition of 600 .mu.l
0.2 M ethanol amine, pH 9.0 after draining the gel by
centrifugation and removal of the supernatant, followed by mixing
at room temperature over night. Non-bound protein was removed by
washing the gel three times with 1 M NaCl (centrifugation followed
by removal of the supernatant) and finally the gel was washed with
250 mM NaCl; 50 mM Tris-HCl, pH 8.0. The enzymatic activity of the
immobilized GrB--H6 C228F was estimated by weighing out drained gel
matrix, mixing with 300 .mu.l of a substrate solution containing
100 mM HEPES, pH 7.75; 400 .mu.M Ac--IEPD-pNA (Calbiochem) and then
measure OD.sub.405 nm of the supernatant after a certain time of
incubation to determine .DELTA.OD.sub.405 nm/min per ml of
substrate solution per g of drained gel. The enzymatic activity of
immobilized GrB--H6 C228F was used to determine the coupling
efficiency as a percentage of the calculated enzymatic activity if
all applied enzyme was coupled and active.
[0452] The coupling efficiency is also listed in Table 10, and
there is no significant difference in coupling efficiency between
the two activation levels and the efficiency is also within the
same range for the two protein concentration, so the highest
coupling level is obtained when using a high protein concentration
in the immobilization mixture. It can not be deduced whether
addition of PEG 20000 to the coupling mixture is favourable for the
immobilization and it was not evaluated for the high protein
concentration.
[0453] The stability of the immobilized GrB--H6 C228F against
denaturation with urea and guanidinium chloride (GdmCl) was
determined for the two immobilizations with high protein
concentration (experiment C and F). The gel matrix from experiment
C and F was each aliquoted into three small spin columns and
incubated with either 8 M urea; 0.5 M NaCl; 50 mM Tris-HCl, pH 8.0
(Urea), with 6 M guanidinium chloride; 50 mM Tris-HCl, pH 8.0
(GdmCl), or with 100 mM HEPES, pH 7.75 (HEPES) for 30 minutes at
room temperature before it was washed and equilibrated in 100 mM
HEPES, pH 7.75. The enzymatic activity of the immobilized GrB--H6
C228F was then determined as described above.
[0454] The enzymatic activities obtained are shown below in Table
11. Denaturation with urea seems to be favourable for the
immobilized GrB--H6 C228F as the enzymatic activity is increased
after incubation compared to incubation with the non-denaturing
HEPES buffer, whereas denaturation with guanidinium chloride seems
to have an effect that slightly decreases the enzymatic activity.
TABLE-US-00011 TABLE 11 Experiment Buffer Enzymatic activity.sup.a
C Urea 99 GdmCl 89 HEPES 91 F Urea 104 GdmCl 78 HEPES 90
.sup.a.DELTA.OD.sub.405/min per ml of substrate solution per g of
drained gel
[0455] Functionality of the immobilized GrB--H6 C228F was
demonstrated by cleavage of the fusion proteins H6-TripUB
IQAD.dwnarw.SP and H6-TripUB IQAD.dwnarw.SG prepared as described
in Example 6. Cleavage experiments were performed with 50 .mu.l of
each gel matrix from the six immobilization experiments. The gel
matrix was re-suspended in 200 .mu.l of a buffer containing 100 mM
HEPES, pH 7.75, and was incubated with 100 .mu.l protein solution
of each of the two fusion proteins at room temperature over night
with shaking. Samples of the supernatant were withdrawn and
analyzed by non-reducing SDS-PAGE, see FIG. 20.
[0456] For all twelve cleavage experiments the fusion proteins are
cleaved to give a single product corresponding to TripUbi moiety
from which the H6 fusion partner has been cleaved off. As expected,
slightly more fusion protein is cleaved by the gel matrix from
experiment C and F that should have the highest coupling levels.
The cleavage efficiency was not evaluated.
REFERENCES
[0457] Christensen J. H. et al. (1991), FEBS Letters, 281(1-2):
181-184.
[0458] Nykj.ae butted.r A. et al. (1992), Journal of Biological
Chemistry, 267(21): 14543-14548.
[0459] Holtet T. L. et al. (1997), Protein Science, 6:
1511-1515
[0460] Studier F. W. et al. (1990), Methods in Enzymology, 185:
60-89
[0461] Hochuli E. et al. (1988), Biotechnology, 1321-1325
[0462] Harris et al. (1998), Journal of Biological Chemistry
273(42): 27364-27373.
[0463] Casciola-Rosen et al. (1999), Journal of Experimental
Medicine 190(6): 815-825.
[0464] Thogersen et al. (1994), International Patent Application WO
9418227
[0465] Sun J. et al. (2001), Journal of Biological Chemistry
276(18): 15177-15184.
Sequence CWU 1
1
57 1 243 PRT Artificial pro-IEGR-GrB-H6 1 Met Gly Ser Ile Glu Gly
Arg Ile Ile Gly Gly His Glu Ala Lys Pro 1 5 10 15 His Ser Arg Pro
Tyr Met Ala Tyr Leu Met Ile Trp Asp Gln Lys Ser 20 25 30 Leu Lys
Arg Cys Gly Gly Phe Leu Ile Gln Asp Asp Phe Val Leu Thr 35 40 45
Ala Ala His Cys Trp Gly Ser Ser Ile Asn Val Thr Leu Gly Ala His 50
55 60 Asn Ile Lys Glu Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys
Arg 65 70 75 80 Pro Ile Pro His Pro Ala Tyr Asn Pro Lys Asn Phe Ser
Asn Asp Ile 85 90 95 Met Leu Leu Gln Leu Glu Arg Lys Ala Lys Arg
Thr Arg Ala Val Gln 100 105 110 Pro Leu Arg Leu Pro Ser Asn Lys Ala
Gln Val Lys Pro Gly Gln Thr 115 120 125 Cys Ser Val Ala Gly Trp Gly
Gln Thr Ala Pro Leu Gly Lys His Ser 130 135 140 His Thr Leu Gln Glu
Val Lys Met Thr Val Gln Glu Asp Arg Lys Cys 145 150 155 160 Glu Ser
Asp Leu Arg His Tyr Tyr Asp Ser Thr Ile Glu Leu Cys Val 165 170 175
Gly Asp Pro Glu Ile Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly 180
185 190 Pro Leu Val Cys Asn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly
Arg 195 200 205 Asn Asn Gly Met Pro Pro Arg Ala Cys Thr Lys Val Ser
Ser Phe Val 210 215 220 His Trp Ile Lys Lys Thr Met Lys Arg Tyr Leu
Asn Ser His His His 225 230 235 240 His His His 2 243 PRT
Artificial pro-IEPD-GrB-H6 2 Met Gly Ser Ile Glu Pro Asp Ile Ile
Gly Gly His Glu Ala Lys Pro 1 5 10 15 His Ser Arg Pro Tyr Met Ala
Tyr Leu Met Ile Trp Asp Gln Lys Ser 20 25 30 Leu Lys Arg Cys Gly
Gly Phe Leu Ile Gln Asp Asp Phe Val Leu Thr 35 40 45 Ala Ala His
Cys Trp Gly Ser Ser Ile Asn Val Thr Leu Gly Ala His 50 55 60 Asn
Ile Lys Glu Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys Arg 65 70
75 80 Pro Ile Pro His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp
Ile 85 90 95 Met Leu Leu Gln Leu Glu Arg Lys Ala Lys Arg Thr Arg
Ala Val Gln 100 105 110 Pro Leu Arg Leu Pro Ser Asn Lys Ala Gln Val
Lys Pro Gly Gln Thr 115 120 125 Cys Ser Val Ala Gly Trp Gly Gln Thr
Ala Pro Leu Gly Lys His Ser 130 135 140 His Thr Leu Gln Glu Val Lys
Met Thr Val Gln Glu Asp Arg Lys Cys 145 150 155 160 Glu Ser Asp Leu
Arg His Tyr Tyr Asp Ser Thr Ile Glu Leu Cys Val 165 170 175 Gly Asp
Pro Glu Ile Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly 180 185 190
Pro Leu Val Cys Asn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg 195
200 205 Asn Asn Gly Met Pro Pro Arg Ala Cys Thr Lys Val Ser Ser Phe
Val 210 215 220 His Trp Ile Lys Lys Thr Met Lys Arg Tyr Leu Asn Ser
His His His 225 230 235 240 His His His 3 243 PRT Artificial
pro-IEAD-GrB-H6 3 Met Gly Ser Ile Glu Ala Asp Ile Ile Gly Gly His
Glu Ala Lys Pro 1 5 10 15 His Ser Arg Pro Tyr Met Ala Tyr Leu Met
Ile Trp Asp Gln Lys Ser 20 25 30 Leu Lys Arg Cys Gly Gly Phe Leu
Ile Gln Asp Asp Phe Val Leu Thr 35 40 45 Ala Ala His Cys Trp Gly
Ser Ser Ile Asn Val Thr Leu Gly Ala His 50 55 60 Asn Ile Lys Glu
Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys Arg 65 70 75 80 Pro Ile
Pro His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp Ile 85 90 95
Met Leu Leu Gln Leu Glu Arg Lys Ala Lys Arg Thr Arg Ala Val Gln 100
105 110 Pro Leu Arg Leu Pro Ser Asn Lys Ala Gln Val Lys Pro Gly Gln
Thr 115 120 125 Cys Ser Val Ala Gly Trp Gly Gln Thr Ala Pro Leu Gly
Lys His Ser 130 135 140 His Thr Leu Gln Glu Val Lys Met Thr Val Gln
Glu Asp Arg Lys Cys 145 150 155 160 Glu Ser Asp Leu Arg His Tyr Tyr
Asp Ser Thr Ile Glu Leu Cys Val 165 170 175 Gly Asp Pro Glu Ile Lys
Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly 180 185 190 Pro Leu Val Cys
Asn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg 195 200 205 Asn Asn
Gly Met Pro Pro Arg Ala Cys Thr Lys Val Ser Ser Phe Val 210 215 220
His Trp Ile Lys Lys Thr Met Lys Arg Tyr Leu Asn Ser His His His 225
230 235 240 His His His 4 243 PRT Artificial pro-IEPD-GrB-H6 C228S
4 Met Gly Ser Ile Glu Pro Asp Ile Ile Gly Gly His Glu Ala Lys Pro 1
5 10 15 His Ser Arg Pro Tyr Met Ala Tyr Leu Met Ile Trp Asp Gln Lys
Ser 20 25 30 Leu Lys Arg Cys Gly Gly Phe Leu Ile Gln Asp Asp Phe
Val Leu Thr 35 40 45 Ala Ala His Cys Trp Gly Ser Ser Ile Asn Val
Thr Leu Gly Ala His 50 55 60 Asn Ile Lys Glu Gln Glu Pro Thr Gln
Gln Phe Ile Pro Val Lys Arg 65 70 75 80 Pro Ile Pro His Pro Ala Tyr
Asn Pro Lys Asn Phe Ser Asn Asp Ile 85 90 95 Met Leu Leu Gln Leu
Glu Arg Lys Ala Lys Arg Thr Arg Ala Val Gln 100 105 110 Pro Leu Arg
Leu Pro Ser Asn Lys Ala Gln Val Lys Pro Gly Gln Thr 115 120 125 Cys
Ser Val Ala Gly Trp Gly Gln Thr Ala Pro Leu Gly Lys His Ser 130 135
140 His Thr Leu Gln Glu Val Lys Met Thr Val Gln Glu Asp Arg Lys Cys
145 150 155 160 Glu Ser Asp Leu Arg His Tyr Tyr Asp Ser Thr Ile Glu
Leu Cys Val 165 170 175 Gly Asp Pro Glu Ile Lys Lys Thr Ser Phe Lys
Gly Asp Ser Gly Gly 180 185 190 Pro Leu Val Cys Asn Lys Val Ala Gln
Gly Ile Val Ser Tyr Gly Arg 195 200 205 Asn Asn Gly Met Pro Pro Arg
Ala Ser Thr Lys Val Ser Ser Phe Val 210 215 220 His Trp Ile Lys Lys
Thr Met Lys Arg Tyr Leu Asn Ser His His His 225 230 235 240 His His
His 5 243 PRT Artificial pro-IEPD-GrB-H6 C228A 5 Met Gly Ser Ile
Glu Pro Asp Ile Ile Gly Gly His Glu Ala Lys Pro 1 5 10 15 His Ser
Arg Pro Tyr Met Ala Tyr Leu Met Ile Trp Asp Gln Lys Ser 20 25 30
Leu Lys Arg Cys Gly Gly Phe Leu Ile Gln Asp Asp Phe Val Leu Thr 35
40 45 Ala Ala His Cys Trp Gly Ser Ser Ile Asn Val Thr Leu Gly Ala
His 50 55 60 Asn Ile Lys Glu Gln Glu Pro Thr Gln Gln Phe Ile Pro
Val Lys Arg 65 70 75 80 Pro Ile Pro His Pro Ala Tyr Asn Pro Lys Asn
Phe Ser Asn Asp Ile 85 90 95 Met Leu Leu Gln Leu Glu Arg Lys Ala
Lys Arg Thr Arg Ala Val Gln 100 105 110 Pro Leu Arg Leu Pro Ser Asn
Lys Ala Gln Val Lys Pro Gly Gln Thr 115 120 125 Cys Ser Val Ala Gly
Trp Gly Gln Thr Ala Pro Leu Gly Lys His Ser 130 135 140 His Thr Leu
Gln Glu Val Lys Met Thr Val Gln Glu Asp Arg Lys Cys 145 150 155 160
Glu Ser Asp Leu Arg His Tyr Tyr Asp Ser Thr Ile Glu Leu Cys Val 165
170 175 Gly Asp Pro Glu Ile Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly
Gly 180 185 190 Pro Leu Val Cys Asn Lys Val Ala Gln Gly Ile Val Ser
Tyr Gly Arg 195 200 205 Asn Asn Gly Met Pro Pro Arg Ala Ala Thr Lys
Val Ser Ser Phe Val 210 215 220 His Trp Ile Lys Lys Thr Met Lys Arg
Tyr Leu Asn Ser His His His 225 230 235 240 His His His 6 243 PRT
Artificial pro-IEPD-GrB-H6 C228T 6 Met Gly Ser Ile Glu Pro Asp Ile
Ile Gly Gly His Glu Ala Lys Pro 1 5 10 15 His Ser Arg Pro Tyr Met
Ala Tyr Leu Met Ile Trp Asp Gln Lys Ser 20 25 30 Leu Lys Arg Cys
Gly Gly Phe Leu Ile Gln Asp Asp Phe Val Leu Thr 35 40 45 Ala Ala
His Cys Trp Gly Ser Ser Ile Asn Val Thr Leu Gly Ala His 50 55 60
Asn Ile Lys Glu Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys Arg 65
70 75 80 Pro Ile Pro His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn
Asp Ile 85 90 95 Met Leu Leu Gln Leu Glu Arg Lys Ala Lys Arg Thr
Arg Ala Val Gln 100 105 110 Pro Leu Arg Leu Pro Ser Asn Lys Ala Gln
Val Lys Pro Gly Gln Thr 115 120 125 Cys Ser Val Ala Gly Trp Gly Gln
Thr Ala Pro Leu Gly Lys His Ser 130 135 140 His Thr Leu Gln Glu Val
Lys Met Thr Val Gln Glu Asp Arg Lys Cys 145 150 155 160 Glu Ser Asp
Leu Arg His Tyr Tyr Asp Ser Thr Ile Glu Leu Cys Val 165 170 175 Gly
Asp Pro Glu Ile Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly 180 185
190 Pro Leu Val Cys Asn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg
195 200 205 Asn Asn Gly Met Pro Pro Arg Ala Thr Thr Lys Val Ser Ser
Phe Val 210 215 220 His Trp Ile Lys Lys Thr Met Lys Arg Tyr Leu Asn
Ser His His His 225 230 235 240 His His His 7 243 PRT Artificial
pro-IEPD-GrB-H6 C228V 7 Met Gly Ser Ile Glu Pro Asp Ile Ile Gly Gly
His Glu Ala Lys Pro 1 5 10 15 His Ser Arg Pro Tyr Met Ala Tyr Leu
Met Ile Trp Asp Gln Lys Ser 20 25 30 Leu Lys Arg Cys Gly Gly Phe
Leu Ile Gln Asp Asp Phe Val Leu Thr 35 40 45 Ala Ala His Cys Trp
Gly Ser Ser Ile Asn Val Thr Leu Gly Ala His 50 55 60 Asn Ile Lys
Glu Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys Arg 65 70 75 80 Pro
Ile Pro His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp Ile 85 90
95 Met Leu Leu Gln Leu Glu Arg Lys Ala Lys Arg Thr Arg Ala Val Gln
100 105 110 Pro Leu Arg Leu Pro Ser Asn Lys Ala Gln Val Lys Pro Gly
Gln Thr 115 120 125 Cys Ser Val Ala Gly Trp Gly Gln Thr Ala Pro Leu
Gly Lys His Ser 130 135 140 His Thr Leu Gln Glu Val Lys Met Thr Val
Gln Glu Asp Arg Lys Cys 145 150 155 160 Glu Ser Asp Leu Arg His Tyr
Tyr Asp Ser Thr Ile Glu Leu Cys Val 165 170 175 Gly Asp Pro Glu Ile
Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly 180 185 190 Pro Leu Val
Cys Asn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg 195 200 205 Asn
Asn Gly Met Pro Pro Arg Ala Val Thr Lys Val Ser Ser Phe Val 210 215
220 His Trp Ile Lys Lys Thr Met Lys Arg Tyr Leu Asn Ser His His His
225 230 235 240 His His His 8 243 PRT Artificial pro-IEPD-GrB-H6
C228F 8 Met Gly Ser Ile Glu Pro Asp Ile Ile Gly Gly His Glu Ala Lys
Pro 1 5 10 15 His Ser Arg Pro Tyr Met Ala Tyr Leu Met Ile Trp Asp
Gln Lys Ser 20 25 30 Leu Lys Arg Cys Gly Gly Phe Leu Ile Gln Asp
Asp Phe Val Leu Thr 35 40 45 Ala Ala His Cys Trp Gly Ser Ser Ile
Asn Val Thr Leu Gly Ala His 50 55 60 Asn Ile Lys Glu Gln Glu Pro
Thr Gln Gln Phe Ile Pro Val Lys Arg 65 70 75 80 Pro Ile Pro His Pro
Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp Ile 85 90 95 Met Leu Leu
Gln Leu Glu Arg Lys Ala Lys Arg Thr Arg Ala Val Gln 100 105 110 Pro
Leu Arg Leu Pro Ser Asn Lys Ala Gln Val Lys Pro Gly Gln Thr 115 120
125 Cys Ser Val Ala Gly Trp Gly Gln Thr Ala Pro Leu Gly Lys His Ser
130 135 140 His Thr Leu Gln Glu Val Lys Met Thr Val Gln Glu Asp Arg
Lys Cys 145 150 155 160 Glu Ser Asp Leu Arg His Tyr Tyr Asp Ser Thr
Ile Glu Leu Cys Val 165 170 175 Gly Asp Pro Glu Ile Lys Lys Thr Ser
Phe Lys Gly Asp Ser Gly Gly 180 185 190 Pro Leu Val Cys Asn Lys Val
Ala Gln Gly Ile Val Ser Tyr Gly Arg 195 200 205 Asn Asn Gly Met Pro
Pro Arg Ala Phe Thr Lys Val Ser Ser Phe Val 210 215 220 His Trp Ile
Lys Lys Thr Met Lys Arg Tyr Leu Asn Ser His His His 225 230 235 240
His His His 9 46 DNA Artificial H6 C-term fw 9 catggacgga
agcttgaatt cacatcacca tcaccatcac taacgc 46 10 46 DNA Artificial H6
C-term rev 10 aattgcgtta gtgatggtga tggtgatgtg aattcaagct tccgct 46
11 40 DNA Artificial GrBfw primer 11 catgggatcc atcgagggta
ggatcatcgg gggacatgag 40 12 38 DNA Artificial GrBrev EcoRI primer
12 gcgtgaattc aggtaccgtt tcatggtttt ctttatcc 38 13 715 DNA
Artificial GrB EcoRI fragment 13 catgggatcc atcgagggta ggatcatcgg
gggacatgag gccaagcccc actcccgccc 60 ctacatggct tatcttatga
tctgggatca gaagtctctg aagaggtgcg gtggcttcct 120 gatacaagac
gacttcgtgc tgacagctgc tcactgttgg ggaagctcca taaatgtcac 180
cttgggggcc cacaatatca aagaacagga gccgacccag cagtttatcc ctgtgaaaag
240 acccatcccc catccagcct ataatcctaa gaacttctcc aacgacatca
tgctactgca 300 gctggagaga aaggccaagc ggaccagagc tgtgcagccc
ctcaggctac ctagcaacaa 360 ggcccaggtg aagccagggc agacatgcag
tgtggccggc tgggggcaga cggcccccct 420 gggaaaacac tcacacacac
tacaagaggt gaagatgaca gtgcaggaag atcgaaagtg 480 cgaatctgac
ttacgccatt attacgacag taccattgag ttgtgcgtgg gggacccaga 540
gattaaaaag acttccttta agggggactc tggaggccct cttgtgtgta acaaggtggc
600 ccagggcatt gtctcctatg gacgaaacaa tggcatgcct ccacgagcct
gcaccaaagt 660 ctcaagcttt gtacactgga taaagaaaac catgaaacgg
tacctgaatt cacgc 715 14 33 DNA Artificial GrB GR-PD fw 14
tccatcgagc cggatatcat cgggggacat gag 33 15 34 DNA Artificial GrB
GR-PD rev 15 ccccgatgat atccggctcg atggatccca tatg 34 16 33 DNA
Artificial GrB GR-AD fw 16 tccatcgagg ctgatatcat cgggggacat gag 33
17 34 DNA Artificial GrB GR-AD rev 17 ccccgatgat atcagcctcg
atggatccca tatg 34 18 27 DNA Artificial GrB SAT fw 18 tccacgagca
dccaccaaag tctcaag 27 19 26 DNA Artificial GrB SAT rev 19
agactttggt gghggctcgt ggaggc 26 20 27 DNA Artificial GrB VF fw 20
tccacgagcc ktcaccaaag tctcaag 27 21 26 DNA Artificial GrB VF rev 21
agactttggt gamggctcgt ggaggc 26 22 150 PRT Artificial H6-TripUB
IEPDSP 22 Met Gly Ser His His His His His His Gly Ser Gly Ser Gly
Ser Ile 1 5 10 15 Glu Pro Asp Ser Pro Gly Thr Glu Pro Pro Thr Gln
Lys Pro Lys Lys 20 25 30 Ile Val Asn Ala Lys Lys Asp Val Val Asn
Thr Lys Met Phe Glu Glu 35 40 45 Leu Lys Ser Arg Leu Asp Thr Leu
Ala Gln Glu Val Ala Leu Leu Lys 50 55 60 Glu Gln Gln Ala Leu Gln
Thr Val Gly Ser Gln Ile Phe Val Lys Thr 65 70 75 80 Leu Thr Gly Lys
Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile 85 90 95 Glu Asn
Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp 100 105 110
Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr 115
120 125 Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val
Leu 130 135 140 Arg Leu Arg Gly Gly Ser 145 150 23 338 PRT
Artificial H6-IEPD-RAP 23 Met Gly Ser His His His His His His Gly
Ser Ile Glu Pro Asp Tyr 1
5 10 15 Ser Arg Glu Lys Asn Gln Pro Lys Pro Ser Pro Lys Arg Glu Ser
Gly 20 25 30 Glu Glu Phe Arg Met Glu Lys Leu Asn Gln Leu Trp Glu
Lys Ala Gln 35 40 45 Arg Leu His Leu Pro Pro Val Arg Leu Ala Glu
Leu His Ala Asp Leu 50 55 60 Lys Ile Gln Glu Arg Asp Glu Leu Ala
Trp Lys Lys Leu Lys Leu Asp 65 70 75 80 Gly Leu Asp Glu Asp Gly Glu
Lys Glu Ala Arg Leu Ile Arg Asn Leu 85 90 95 Asn Val Ile Leu Ala
Lys Tyr Gly Leu Asp Gly Lys Lys Asp Ala Arg 100 105 110 Gln Val Thr
Ser Asn Ser Leu Ser Gly Thr Gln Glu Asp Gly Leu Asp 115 120 125 Asp
Pro Arg Leu Glu Lys Leu Trp His Lys Ala Lys Thr Ser Gly Lys 130 135
140 Phe Ser Gly Glu Glu Leu Asp Lys Leu Trp Arg Glu Phe Leu His His
145 150 155 160 Lys Glu Lys Val His Glu Tyr Asn Val Leu Leu Glu Thr
Leu Ser Arg 165 170 175 Thr Glu Glu Ile His Glu Asn Val Ile Ser Pro
Ser Asp Leu Ser Asp 180 185 190 Ile Lys Gly Ser Val Leu His Ser Arg
His Thr Glu Leu Lys Glu Lys 195 200 205 Leu Arg Ser Ile Asn Gln Gly
Leu Asp Arg Leu Arg Arg Val Ser His 210 215 220 Gln Gly Tyr Ser Thr
Glu Ala Glu Phe Glu Glu Pro Arg Val Ile Asp 225 230 235 240 Leu Trp
Asp Leu Ala Gln Ser Ala Asn Leu Thr Asp Lys Glu Leu Glu 245 250 255
Ala Phe Arg Glu Glu Leu Lys His Phe Glu Ala Lys Ile Glu Lys His 260
265 270 Asn His Tyr Gln Lys Gln Leu Glu Ile Ala His Glu Lys Leu Arg
His 275 280 285 Ala Glu Ser Val Gly Asp Gly Glu Arg Val Ser Arg Ser
Arg Glu Lys 290 295 300 His Ala Leu Leu Glu Gly Arg Thr Lys Glu Leu
Gly Tyr Thr Val Lys 305 310 315 320 Lys His Leu Gln Asp Leu Ser Gly
Arg Ile Ser Arg Ala Arg His Asn 325 330 335 Glu Leu 24 336 PRT
Artificial H6Ubi-IEPD-ApoA1 24 Met Gly Ser His His His His His His
Gly Ser Gln Ile Phe Val Lys 1 5 10 15 Thr Leu Thr Gly Lys Thr Ile
Thr Leu Glu Val Glu Pro Ser Asp Thr 20 25 30 Ile Glu Asn Val Lys
Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro 35 40 45 Asp Gln Gln
Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg 50 55 60 Thr
Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val 65 70
75 80 Leu Arg Leu Arg Gly Gly Ser Ile Glu Pro Asp Gly Gly Asp Glu
Pro 85 90 95 Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr
Val Tyr Val 100 105 110 Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val
Ser Gln Phe Glu Gly 115 120 125 Ser Ala Leu Gly Lys Gln Leu Asn Leu
Lys Leu Leu Asp Asn Trp Asp 130 135 140 Ser Val Thr Ser Thr Phe Ser
Lys Leu Arg Glu Gln Leu Gly Pro Val 145 150 155 160 Thr Gln Glu Phe
Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg 165 170 175 Gln Glu
Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro 180 185 190
Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr 195
200 205 Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala
Arg 210 215 220 Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu
Gly Glu Glu 225 230 235 240 Met Arg Asp Arg Ala Arg Ala His Val Asp
Ala Leu Arg Thr His Leu 245 250 255 Ala Pro Tyr Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala Arg Leu Glu 260 265 270 Ala Leu Lys Glu Asn Gly
Gly Ala Arg Leu Ala Glu Tyr His Ala Lys 275 280 285 Ala Thr Glu His
Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu 290 295 300 Glu Asp
Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val 305 310 315
320 Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln
325 330 335 25 197 PRT Artificial H6-IEPD-TN123 25 Met Gly Ser His
His His His His His Gly Ser Ile Glu Pro Asp Gly 1 5 10 15 Glu Pro
Pro Thr Gln Lys Pro Lys Lys Ile Val Asn Ala Lys Lys Asp 20 25 30
Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser Arg Leu Asp Thr 35
40 45 Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln Gln Ala Leu Gln
Thr 50 55 60 Val Cys Leu Lys Gly Thr Lys Val His Met Lys Cys Phe
Leu Ala Phe 65 70 75 80 Thr Gln Thr Lys Thr Phe His Glu Ala Ser Glu
Asp Cys Ile Ser Arg 85 90 95 Gly Gly Thr Leu Ser Thr Pro Gln Thr
Gly Ser Glu Asn Asp Ala Leu 100 105 110 Tyr Glu Tyr Leu Arg Gln Ser
Val Gly Asn Glu Ala Glu Ile Trp Leu 115 120 125 Gly Leu Asn Asp Met
Ala Ala Glu Gly Thr Trp Val Asp Met Thr Gly 130 135 140 Ala Arg Ile
Ala Tyr Lys Asn Trp Glu Thr Glu Ile Thr Ala Gln Pro 145 150 155 160
Asp Gly Gly Lys Thr Glu Asn Cys Ala Val Leu Ser Gly Ala Ala Asn 165
170 175 Gly Lys Trp Phe Asp Lys Arg Cys Arg Asp Gln Leu Pro Tyr Ile
Cys 180 185 190 Gln Phe Gly Ile Val 195 26 150 PRT Artificial
H6-TripUB IQADSP 26 Met Gly Ser His His His His His His Gly Ser Gly
Ser Gly Ser Ile 1 5 10 15 Gln Ala Asp Ser Pro Gly Thr Glu Pro Pro
Thr Gln Lys Pro Lys Lys 20 25 30 Ile Val Asn Ala Lys Lys Asp Val
Val Asn Thr Lys Met Phe Glu Glu 35 40 45 Leu Lys Ser Arg Leu Asp
Thr Leu Ala Gln Glu Val Ala Leu Leu Lys 50 55 60 Glu Gln Gln Ala
Leu Gln Thr Val Gly Ser Gln Ile Phe Val Lys Thr 65 70 75 80 Leu Thr
Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile 85 90 95
Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp 100
105 110 Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg
Thr 115 120 125 Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His
Leu Val Leu 130 135 140 Arg Leu Arg Gly Gly Ser 145 150 27 150 PRT
Artificial H6-TripUB IQADSG 27 Met Gly Ser His His His His His His
Gly Ser Gly Ser Gly Ser Ile 1 5 10 15 Gln Ala Asp Ser Gly Gly Thr
Glu Pro Pro Thr Gln Lys Pro Lys Lys 20 25 30 Ile Val Asn Ala Lys
Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu 35 40 45 Leu Lys Ser
Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys 50 55 60 Glu
Gln Gln Ala Leu Gln Thr Val Gly Ser Gln Ile Phe Val Lys Thr 65 70
75 80 Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr
Ile 85 90 95 Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile
Pro Pro Asp 100 105 110 Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu
Glu Asp Gly Arg Thr 115 120 125 Leu Ser Asp Tyr Asn Ile Gln Lys Glu
Ser Thr Leu His Leu Val Leu 130 135 140 Arg Leu Arg Gly Gly Ser 145
150 28 150 PRT Artificial H6-TripUB VGPDSP 28 Met Gly Ser His His
His His His His Gly Ser Gly Ser Gly Ser Val 1 5 10 15 Gly Pro Asp
Ser Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys 20 25 30 Ile
Val Asn Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu 35 40
45 Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys
50 55 60 Glu Gln Gln Ala Leu Gln Thr Val Gly Ser Gln Ile Phe Val
Lys Thr 65 70 75 80 Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro
Ser Asp Thr Ile 85 90 95 Glu Asn Val Lys Ala Lys Ile Gln Asp Lys
Glu Gly Ile Pro Pro Asp 100 105 110 Gln Gln Arg Leu Ile Phe Ala Gly
Lys Gln Leu Glu Asp Gly Arg Thr 115 120 125 Leu Ser Asp Tyr Asn Ile
Gln Lys Glu Ser Thr Leu His Leu Val Leu 130 135 140 Arg Leu Arg Gly
Gly Ser 145 150 29 150 PRT Artificial H6-TripUB VGPDFG 29 Met Gly
Ser His His His His His His Gly Ser Gly Ser Gly Ser Val 1 5 10 15
Gly Pro Asp Phe Gly Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys 20
25 30 Ile Val Asn Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu
Glu 35 40 45 Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln Glu Val Ala
Leu Leu Lys 50 55 60 Glu Gln Gln Ala Leu Gln Thr Val Gly Ser Gln
Ile Phe Val Lys Thr 65 70 75 80 Leu Thr Gly Lys Thr Ile Thr Leu Glu
Val Glu Pro Ser Asp Thr Ile 85 90 95 Glu Asn Val Lys Ala Lys Ile
Gln Asp Lys Glu Gly Ile Pro Pro Asp 100 105 110 Gln Gln Arg Leu Ile
Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr 115 120 125 Leu Ser Asp
Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu 130 135 140 Arg
Leu Arg Gly Gly Ser 145 150 30 143 PRT Artificial H6-TripUB IEPDTQ
30 Met Gly Ser His His His His His His Gly Ser Gly Ser Gly Ser Ile
1 5 10 15 Glu Pro Asp Thr Gln Lys Pro Lys Lys Ile Val Asn Ala Lys
Lys Asp 20 25 30 Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys Ser
Arg Leu Asp Thr 35 40 45 Leu Ala Gln Glu Val Ala Leu Leu Lys Glu
Gln Gln Ala Leu Gln Thr 50 55 60 Val Gly Ser Gln Ile Phe Val Lys
Thr Leu Thr Gly Lys Thr Ile Thr 65 70 75 80 Leu Glu Val Glu Pro Ser
Asp Thr Ile Glu Asn Val Lys Ala Lys Ile 85 90 95 Gln Asp Lys Glu
Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala 100 105 110 Gly Lys
Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln 115 120 125
Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Ser 130 135
140 31 137 PRT Artificial H6-TripUB IEPDIV 31 Met Gly Ser His His
His His His His Gly Ser Gly Ser Gly Ser Ile 1 5 10 15 Glu Pro Asp
Ile Val Asn Ala Lys Lys Asp Val Val Asn Thr Lys Met 20 25 30 Phe
Glu Glu Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln Glu Val Ala 35 40
45 Leu Leu Lys Glu Gln Gln Ala Leu Gln Thr Val Gly Ser Gln Ile Phe
50 55 60 Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu
Pro Ser 65 70 75 80 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp
Lys Glu Gly Ile 85 90 95 Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala
Gly Lys Gln Leu Glu Asp 100 105 110 Gly Arg Thr Leu Ser Asp Tyr Asn
Ile Gln Lys Glu Ser Thr Leu His 115 120 125 Leu Val Leu Arg Leu Arg
Gly Gly Ser 130 135 32 150 PRT Artificial H6-TripUB IEPDEP 32 Met
Gly Ser His His His His His His Gly Ser Gly Ser Gly Ser Ile 1 5 10
15 Glu Pro Asp Glu Pro Gly Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys
20 25 30 Ile Val Asn Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe
Glu Glu 35 40 45 Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln Glu Val
Ala Leu Leu Lys 50 55 60 Glu Gln Gln Ala Leu Gln Thr Val Gly Ser
Gln Ile Phe Val Lys Thr 65 70 75 80 Leu Thr Gly Lys Thr Ile Thr Leu
Glu Val Glu Pro Ser Asp Thr Ile 85 90 95 Glu Asn Val Lys Ala Lys
Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp 100 105 110 Gln Gln Arg Leu
Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr 115 120 125 Leu Ser
Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu 130 135 140
Arg Leu Arg Gly Gly Ser 145 150 33 150 PRT Artificial H6-TripUB
IEPDEG 33 Met Gly Ser His His His His His His Gly Ser Gly Ser Gly
Ser Ile 1 5 10 15 Glu Pro Asp Glu Gly Gly Thr Glu Pro Pro Thr Gln
Lys Pro Lys Lys 20 25 30 Ile Val Asn Ala Lys Lys Asp Val Val Asn
Thr Lys Met Phe Glu Glu 35 40 45 Leu Lys Ser Arg Leu Asp Thr Leu
Ala Gln Glu Val Ala Leu Leu Lys 50 55 60 Glu Gln Gln Ala Leu Gln
Thr Val Gly Ser Gln Ile Phe Val Lys Thr 65 70 75 80 Leu Thr Gly Lys
Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile 85 90 95 Glu Asn
Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp 100 105 110
Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr 115
120 125 Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val
Leu 130 135 140 Arg Leu Arg Gly Gly Ser 145 150 34 37 DNA
Artificial TripUB GrB fw primer 34 gtggatccat cgagcctgac tctcctggta
ccgagcc 37 35 38 DNA Artificial TripUB GrB rev primer 35 ggtaccagga
gagtcaggct cgatggatcc actaccac 38 36 34 DNA Artificial RAP GrB fw
primer 36 cggatccatc gagcctgact actcgcggga gaag 34 37 34 DNA
Artificial RAP GrB rev primer 37 cccgcgagta gtcaggctcg atggatccgt
gatg 34 38 42 DNA Artificial Mut-GrB fw 38 cgtggtggat ccatcgagcc
ggacggtgga gatgaacccc cc 42 39 42 DNA Artificial Mut-GrB rw 39
ggggggttca tctccaccgt ccggctcgat ggatccacca cg 42 40 33 DNA
Artificial TN GrB fw primer 40 ggatccatcg agcctgacgg cgagccacca acc
33 41 33 DNA Artificial TN GrB rev primer 41 ggctcgccgt caggctcgat
ggatccgtga tgg 33 42 33 DNA Artificial PC7TripUB GR-AD fw 42
ggatccatcc aggcagactc tcctggtacc gag 33 43 34 DNA Artificial
PC7TripUB GR-AD rev 43 gtaccaggag agtctgcctg gatggatcca ctac 34 44
37 DNA Artificial PC7TripUB P-G fw 44 ggatccatcc aggcagactc
tggtggtacc gagccac 37 45 38 DNA Artificial PC7TripUB P-G rev 45
ctcggtacca ccagagtctg cctggatgga tccactac 38 46 34 DNA Artificial
DNATrip IE-VG fw 46 gtagtggatc agtcgggcct gactctcctg gtac 34 47 34
DNA Artificial DNATrip IE-VG rev 47 gagagtcagg cccgactgat
ccactaccac tacc 34 48 31 DNA Artificial DNATrip SP-FG fw 48
ggcctgactt tggtggtacc gagccaccaa c 31 49 31 DNA Artificial DNATrip
SP-FG rev 49 ggctcggtac caccaaagtc aggcccgact g 31 50 52 DNA
Artificial Trip IEPD-TQ 50 gggaaaggat ccatcgagcc tgacacccag
aagcccaaga agattgtaaa tg 52 51 54 DNA Artificial Trip IEPD-IV 51
gggaaaggat ccatcgagcc tgacattgta aatgccaaga aagatgttgt gaac 54 52
39 DNA Artificial UB3 52 cgcaagcttg catgcttagg atccaccacg aagtctcaa
39 53 29 DNA Artificial TripUB EP fw 53 cgagcctgac gagcctggta
ccgagccac 29 54 30 DNA Artificial TripUB EP rev 54 cggtaccagg
ctcgtcaggc tcgatggatc 30 55 29 DNA Artificial TripUB EG fw 55
cctgacgagg gtggtaccga gccaccaac 29 56 29 DNA Artificial TripUB EG
rev 56 gctcggtacc accctcgtca ggctcgatg
29 57 227 PRT Artificial GrB variant C228F 57 Ile Ile Gly Gly His
Glu Ala Lys Pro His Ser Arg Pro Tyr Met Ala 1 5 10 15 Tyr Leu Met
Ile Trp Asp Gln Lys Ser Leu Lys Arg Cys Gly Gly Phe 20 25 30 Leu
Ile Gln Asp Asp Phe Val Leu Thr Ala Ala His Cys Trp Gly Ser 35 40
45 Ser Ile Asn Val Thr Leu Gly Ala His Asn Ile Lys Glu Gln Glu Pro
50 55 60 Thr Gln Gln Phe Ile Pro Val Lys Arg Pro Ile Pro His Pro
Ala Tyr 65 70 75 80 Asn Pro Lys Asn Phe Ser Asn Asp Ile Met Leu Leu
Gln Leu Glu Arg 85 90 95 Lys Ala Lys Arg Thr Arg Ala Val Gln Pro
Leu Arg Leu Pro Ser Asn 100 105 110 Lys Ala Gln Val Lys Pro Gly Gln
Thr Cys Ser Val Ala Gly Trp Gly 115 120 125 Gln Thr Ala Pro Leu Gly
Lys His Ser His Thr Leu Gln Glu Val Lys 130 135 140 Met Thr Val Gln
Glu Asp Arg Lys Cys Glu Ser Asp Leu Arg His Tyr 145 150 155 160 Tyr
Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro Glu Ile Lys Lys 165 170
175 Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Lys Val
180 185 190 Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly Met Pro
Pro Arg 195 200 205 Ala Phe Thr Lys Val Ser Ser Phe Val His Trp Ile
Lys Lys Thr Met 210 215 220 Lys Arg Tyr 225
* * * * *
References